DNA for the production of multimeric hemoglobins

ABSTRACT

DNA molecules which encode pseudodimeric globin-like polypeptides with an asymmetric cysteine mutation suitable for crosslinking two tetramers, or which encode pseudooligomeric globin-like polypeptides comprising four or more globin-like domains, are useful in the preparation of multimeric hemoglobin-like proteins.

This application is a continuation of Ser. No. 08/240,712, filed May 9,1994, now U.S. Pat. No. 5,595,903, which is a continuation-in-part ofSer. No. 07/789,179, filed Nov. 8, 1991, now U.S. Pat. No. 5,543,727,which is a continuation-in-part of Ser. No. 07/671,707, filed Apr. 1,1991, now abandoned, which is the national stage of PCT/US90/02654,filed May 10, 1990, which is a continuation-in-part of (a) Looker andHoffman, U.S. Ser. No. 07/374,161, DI-ALPHA AND DI-BETA GLOBIN LIKEPOLYPEPTIDES AND USES THEREFOR, filed Jun. 30, 1989 now abandoned; (b)Stetler and Wagenbach, U.S. Ser. No. 07/379,116, PRODUCTION OF HUMANHEMOGLOBIN BY TRANSFORMED YEAST CELLS, filed Jul. 13, 1989 nowabandoned; and (c) Hoffman, Looker, Rosendahl and Stetler, U.S. Ser. No.07/349,623, POLYCISTRONIC CO-EXPRESSION OF THE ALPHA- AND BETA-GLOBINSAND IN VIVO ASSEMBLY OF BIOLOGICALLY ACTIVE, TETRAMERIC HEMOGLOBIN,filed May 10, 1989 now abandoned; all owned by Somatogen, Inc.

OTHER APPLICATIONS

Hoffman and Nagai, U.S. Ser. No. 07/194,338, filed May 10, 1988, nowU.S. Pat. No. 5,028,588, presently owned by Somatogen, Inc., relates tothe use of low oxygen affinity and other mutant hemoglobins as bloodsubstitutes, and to the expression of alpha and beta globin innonerythroid cells. Hoffman and Nagai, U.S. Ser. No. 07/443,950, filedDec. 1, 1989, now abandoned discloses certain additional dicysteinehemoglobin mutants; it is a continuation-in-part of 07/194,338, now U.S.Pat. No. 5,028,588. Anderson, et al., HEMOGLOBINS AS DRUG DELIVERYAGENTS, Ser. No. 07/789,177, filed Nov. 8, 1991, now abandoned disclosesuse of conjugation of hemoglobins with drugs as a means for delivery ofthe drug to a patient.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is directed to multimeric hemoglobin-like proteinscomposed of two or more pseudotetramers linked together either bygenetic fusion or by chemical crosslinking.

2. Description of the Background Art

A. Structure and Function of Hemoglobin

Hemoglobin (Hgb or HB) is the oxygen-carrying component of blood.Hemoglobin circulates through the bloodstream inside small enucleatecells called erythrocytes (red blood cells). Hemoglobin is a proteinconstructed from four associated polypeptide chains, and bearingprosthetic groups known as hemes. The erythrocyte helps maintainhemoglobin in its reduced, functional form. The heme iron atom issusceptible to oxidation, but may be reduced again by one of two enzymesystems within the erythrocyte, the cytochrome b₅ and glutathionereduction systems.

The structure of hemoglobin is well known. We herewith incorporate byreference the entire text of Bunn and Forget, eds., Hemoglobin:Molecular, Genetic and Clinical Aspects (W. B. Saunders Co.,Philadelphia, Pa.: 1986) and of Fermi and Perutz "Hemoglobin andMyoglobin," in Phillips and Richards, Atlas of Molecular Structures inBiology (Clarendon Press: 1981).

About 92% of the normal adult human hemolysate is Hgb A (designatedalpha2 beta2, because it comprises two alpha and two beta chains). Otherrecognized hemoglobin species are Hgb A₂ (α₂ δ₂), Hgb A_(1a), HgbA_(1b), and Hgb A_(1c), as well as the rare species Hgb F (α₂ gamma₂),Hgb Gower-1 (Zeta₂ epsilon₂) Hgb Gower-2 (alpha₂ epsilon₂), Hgb Portland(Zeta₂ gamma₂), and Hgb H (beta₄) and Hgb Bart (gamma₄). They aredistinguished from Hgb A by a different selection of polypeptide chains.

The primary structure of a polypeptide is defined by its amino acidsequence and by identification of any modifications of the side chainsof the individual amino acids. The amino acid sequences of both thealpha and beta globin polypeptide chains of "normal" human hemoglobin isgiven in Table 1. Many mutant forms are also known; several mutants areidentified in Table 400. The wild-type alpha chain consists of 141 aminoacids. The iron atom of the heme (ferroprotoporphyrin IX) group is boundcovalently to the imidazole of His 87 (the "proximal histidine"). Thewild-type beta chain is 146 residues long and heme is bound to it at His92. Apohemoglobin is the heme-free analogue of hemoglobin; it existspredominantly as the αβ-globin dimer.

Segments of polypeptide chains may be stabilized by folding into one oftwo common conformations, the alpha helix and the beta pleated sheet. Inits native state, about 75% of the hemoglobin molecule is alpha-helical.Alpha-helical segments are separated by segments wherein the chain isless constrained. It is conventional to identify the alpha-helicalsegments of each chain by letters, e.g., the proximal histidine of thealpha chain is F8 (residue 8 of helix F). The non- helical segments areidentified by letter pairs, indicating which helical segments theyconnect. Thus, nonhelical segment BC lies between helix B and helix C.In comparing two variants of a particular hemoglobin chain, it may beenlightening to attempt to align the helical segments when seeking tofind structural homologies. For the amino acid sequence and helicalresidue notation for normal human hemoglobin A_(o) alpha and betachains, see Bunn and Forget, supra, and Table 1 herein.

The tertiary structure of the hemoglobin molecule refers to the stericrelationships of amino acid residues that are far apart in the linearsequence, while quaternary structure refers to the way in which thesubunits (chains) are packed together. The tertiary and quaternarystructure of the hemoglobin molecule have been discerned by X-raydiffraction analysis of hemoglobin crystals, which allows one tocalculate the three-dimensional positions of the very atoms of themolecule.

In its unoxygenated ("deoxy", or "T" for "tense") form, the subunits ofhemoglobin A (alpha1, alpha2, beta1, and beta2) form a tetrahedronhaving a twofold axis of symmetry. The axis runs down a water-filled"central cavity". The subunits interact with one another by means of Vander Waals forces, hydrogen bonds and by ionic interactions (or "saltbridges"). The alphalbetal and alpha2beta2 interfaces remain relativelyfixed during oxygenation. In contrast, there is considerable flux at thealpha1beta2 (and alpha2beta1) interface. In its oxygenated ("oxy", or"R" for "relaxed" form), the intersubunit distances are increased.

The tertiary and quaternary structures of native Oxyhemoglobin anddeoxyhemoglobin are sufficiently well known that almost all of thenonhydrogen atoms can be positioned with an accuracy of 0.5 Å or better.For human deoxyhemoglobin, see Fermi, et al., J. Mol. Biol., 175: 159(1984), and for human oxyhemoglobin, see Shaanan, J. Mol. Biol., 171:31(1983), both incorporated by reference.

Normal hemoglobin has cysteines at beta 93 (F9), beta 112 (G14), andalpha 104 (G11). The latter two positions are deeply buried in both theoxy and deoxy states; they lie near the α₁ β₁ interface. Beta 93,however, in the oxy form is reactive with sulfhydryl reagents.

Native human hemoglobin has been fully reconstituted from separatedheme-free alpha and beta globin and from hemin. Preferably, heme isfirst added to the alpha-globin subunit. The heme-bound alpha globin isthen complexed to the heme-free beta subunit. Finally, heme is added tothe half-filled globin dimer, and tetrameric hemoglobin is obtained.Yip, et al., PNAS (U.S.A.), 74: 64-68 (1977).

The human alpha and beta globin genes reside on chromosomes 16 and 11,respectively. Bunn and Forget, infra at 172. Both genes have been clonedand sequenced, Liebhaber, et al., PNAS 77: 7054-58 (1980) (alpha-globingenomic DNA); Marotta, et al., J. Biol. Chem., 252: 5040-57 (1977) (betaglobin cDNA); Lawn, et al., Cell, 21:647 (1980) (beta globin genomicDNA).

Hemoglobin exhibits cooperative binding of oxygen by the four subunitsof the hemoglobin molecule (two alpha-globins and two beta-globins inthe case of Hgb A), and this cooperativity greatly facilitates efficientoxygen transport. Cooperativity, achieved by the so-called hemehemeinteraction, allows hemoglobin to vary its affinity for oxygen.Hemoglobin reversibly binds up to four moles of oxygen per mole of Hgb.

Oxygen-carrying compounds are frequently compared by means of a deviceknown as an oxygen dissociation curve. This curve is obtained when, fora given oxygen carrier, oxygen saturation or content is graphed againstthe partial pressure of oxygen. For Hgb, the percentage of saturationincreases with partial pressure according to a sigmoid relationship. TheP₅₀ is the partial pressure at which the oxygen-carrying solution ishalf saturated with oxygen. It is thus a measure of oxygen-bindingaffinity; the higher the P₅₀, the more loosely the oxygen is held.

When the oxygen dissociation curve of an oxygen-carrying solution issuch that the P₅₀ is less than that for whole blood, it is said to be"left-shifted."

The oxygen affinity of hemoglobin is lowered by the presence of2,3-diphosphoglycerate (2,3-DPG), chloride ions and hydrogen ions.Respiring tissue releases carbon dioxide into the blood and lowers itspH (i.e. increases the hydrogen ion concentration), thereby causingoxygen to dissociate from hemoglobin and allowing it to diffuse intoindividual cells.

The ability of hemoglobin to alter its oxygen affinity, increasing theefficiency of oxygen transport around the body, is dependent on thepresence of the metabolite 2,3- DPG. Inside the erythrocyte 2,3-DPG ispresent at a concentration nearly as great as that of hemoglobin itself.In the absence of 2,3-DPG "conventional" hemoglobin binds oxygen verytigthly and would release little oxygen to respiring tissue.

Aging erythrocytes release small amounts; of free hemoglobin into theblood plasma where it is rapidly bound by the scavenging proteinhaptoglobin. The hemoglobin-haptoglobin complex is removed from theblood and degraded by the spleen and liver.

Isolated alpha globin chains are monomers; exhibit high oxygen affinitybut of course lack subunit cooperativity. Isolated beta globin chainsaggregate to form a β₄ tetramer (HbH). The β₄ tetramer has a high butnoncooperative oxygen affinity.

B. Blood Substitutes, Generally

It is not always practical to transfuse a patient with donated blood. Inthese situations, use of a red blood cell substitute is desirable. Theproduct must effectively transport O₂, just as do red blood cells.("Plasma expanders", such as dextran and albumin, do not transportoxygen.) The two types of substitutes that have been studied mostextensively are hemoglobin solutions and fluorocarbon emulsions.

It is clear from the above considerations that free native hemoglobin A,injected directly into the bloodstream, would not support efficientoxygen transport about the body. The essential allosteric regulator2,3-DPG is not present in sufficient concentration in the plasma toallow hemoglobin to release much oxygen at venous oxygen tension.

Nonetheless, solutions of conventional hemoglobin have been used as RBCsubstitutes. The classic method of preparing hemoglobin solutionsemploys outdated blood. The red cells are lysed and cellular debris isremoved, leaving what is hopefully "stromal-free hemoglobin" (SFH).

Several basic problems have been observed with this approach. Thesolution must be freed of any toxic components of the red cell membranewithout resorting to cumbersome and tedious procedures which woulddiscourage large-scale production. DeVenuto, "Appraisal of HemoglobinSolution as a Blood Substitute", Surgery, Gynecology and Obstetrics,149: 417-436 (1979).

Second, as expected, such solutions are "left-shifted" (lower P₅₀) ascompared to whole blood. Gould, et al., "The Development of PolymerizedPyridoxylated Hemoglobin Solution as a Red Cell Substitute", Ann. Emerg.Med. 15: 1416-1419 (Dec. 3, 1986). As a result, the oxygen affinity istoo high to unload enough oxygen into the tissues. Benesch and Benesch,Biochem. Biophys. Res. Comm., 26:162-167 (1967).

Third, SFH has only a limited half-life in the circulatory system. Thisis because oxy Hgb partially dissociates into a dimer (αβ) that israpidly cleared from the blood by glomerular filtration and binding tocirculating haptoglobulin. If large amounts of soluble hemoglobin areintroduced into the circulation, glomerular filtration of the dimers maylead to a protein and iron load on the kidney capable of causing renaldamage. Bunn, H. F., et al. (1969) The renal handling of hemoglobin I.Glomerular filtration. J. Exp. Med. 129:909-923; Bunn, H .F., and J. H.Jandl; (1969) The renal handling of hemoglobin II. Catabolism. J. Exp.Med. 129:925-934; Lee, R. L., et al. (1989) Ultrapure, stroma-free,polymerized bovine hemoglobin solution: Evaluation of renal toxicity(blood substitutes) J. Surgical Res. 47:407-411; Feola, M., et al.(1990) Nephrotoxicity of hemoglobin solutions. Biomat. Art. Cell Art.Org., 18(2):233-249; Tam, S. C. and J. T. F. Wong (1988) Impairment ofrenal function by stromafree hemoglobin in rats. J. Lab. Clin. Med.111:189-193.

Finally, SFH has a high colloid osmotic pressure (COD). Thus,administration of SFH in a dose that would have the same oxygen-carryingcapacity as a unit of packed red blood cells is inadvisable, since thehigh osmotic pressure would cause a massive influx of water from thecells into the bloodstream, thus dehydrating the patient's tissues. Thisconsideration limits the dose of SFH to that which provide a finalconcentration of about 6-8 gm Hgb/dl.

In an effort to restore the desired P₅₀, researchers added 2,3-DPG tothe hemoglobin solution. Unfortunately, 2,3- DPG was rapidly eliminatedfrom the circulation. Scientists then turned to other organicphosphates, particularly pyridoxal phosphate. Like 2,3-DPG, thesecompounds stabilized the "T state" of the Hgb by forming a salt bridgebetween the N- termini of the two beta chains. The pyridoxylatedhemoglobin had a P₅₀ of 20-22 torr, as compared to 10 torr for SFH and28 torr for whole blood. While this is an improvement over SFH, thepyridoxylated Hgb remains "high affinity" relative to whole blood.

C. Naturally Occurring Cysteine Substitution Mutants of Hemoglobin(Non-Polymerizing)

There are a few known naturally occurring mutants of human hemoglobin inwhich a cysteine residue is substituted for another residue of normalhemoglobin Ao.

In hemoglobin Nigeria, the mutation is α 81 Ser → Cys; no disulfide isformed. Honig, et al., Blood, 55(1):131-137 (1980). In HemoglobinRainier, an intrasubunit disulfide is formed between the wild typeF9(93)β Cysteine and the cysteine introduced by replacement of the Tyrat HC2(145)β. Greer, et al., Nature New Biology!, 230:261-264 (1971).Hemoglobin Nunobiki (βα141 Arg→Cys) also features a non-polymerizingcysteine substitution. In both Hb Rainier and Hb Nunobiki, the newcysteine residues are on the surface of the tetramer.

D. Naturally Occurring Polymerizing or Polymeric Hemoglobins

Three other human mutants are known which polymerize as a result offormation of intermolecular (first tetramer to second tetramer)disulfide bridges. In Hemoglobin Porto Alegre, the Ser at A6(9)β isreplaced by Cysteine, and since this cysteinyl residue is externallyoriented, spontaneous polymerization occurs, and results in formation ofa dodecamer with three Porto Alegre tetramers linked by disulfide bonds.An octamer has also been made by a 1:1 mixture of Porto Alegrehemoglobin and normal hemoglobin. Tondo, Biochem. Biophys. Acta,342:15-20 (1974); Tondo, An. Acad. Bras. Ci., 59:243-251 (1987).

Hb Mississippi is characterized by a cysteine substitution in place ofSer CD3(44)β. Hemolysates of a patient were subjected to gel filtrationcolumn chromatography, and 48.8% eluted in the void volume. Since themolecular weight exclusion was about 600 kD, this suggested that Hb MSpolymers are composed of ten or more hemoglobin tetramers. Adams, etal., Hemoglobin, 11(5):435-452 (1987).

A β83(EF7)Gly→Cys mutation characterizes Hemoglobin Ta Li. This mutantshowed slow mobility in starch gel electrophoresis, indicating that itwas a polymer.

Polymeric mouse hemoglobins have been reported. In BALB/cJ mice, thereis a reactive cysteinyl residue near the NH₂ -terminal of the beta chain(β-13 in the mouse). This mouse mutant has been compared to HemoglobinPorto Alegre, which likewise has a cysteinyl residue in the A-helix ofthe beta chain. Octamer formation is most common. However, each tetramerhas two extra cysteinyl residues, one on each α-chain, that may reactwith different tetramers; "this explains why aggregates larger thanoctamers occur". Bonaventura and Riggs, Science, 158:800-802 (1967);Riggs, Science, 147:621-623 (1965).

Macaques also exhibit a polymerizing hemoglobin variant. Takenaka, etal., Biopchem. Biophys. Acta, 492:433-444 (1977); ishimoto, et al., J.Anthrop. Soc. Nippon, 83(3):233-243 (1975). This mutant has beencompared to the Ta Li variant in humans.

Both amphibians and reptiles possess polymerizing hemoglobins. Forexample, in the bullfrog, hemoglobin "Component C" polymerizes bydisulfide bond formation between tetramers. This is said to be primarilydependent on cysteinyl residues of the alpha chain. Tam, et al., J.Biol. Chem., 261:8290-94 (1986).

The extracellular hemoglobin of the earthworm (Lumbricus terrestris) hasa complex structure. There are twelve subunits, each being a dimer ofstructure (abcd)₂ where "a", "b", "c", and "d" denote the major hemecontaining chains. The "a", "b", and "c" chains form a disulfide-linkedtrimer. The whole molecule is composed of 192 heme-containing chains and12 non-heme chains, and has a molecular weight of 3800 kDa. Otherinvertebrate hemoglobins are also large multi-subunit proteins.

The brine shrimp Artemia produces three polymeric hemoglobins with ninegenetically fused globin subunits. Manning, et al., Nature, 348:653(1990). These are formed by variable association of two differentsubunit types, α and β of the eight intersubunit linkers, six are 12residues long, one is 11 residues and one is 14 residues.

E. Artificially Crosslinked Hemoglobins (Non-Polymerizing)

The properties of hemoglobin have been altered by specificallychemically crosslinking the alpha chains between the Lys99 of alphal andthe Lys99 of alpha2 Walder, U. S. 4,600,531 and 4,598,064; Snyder, etal., PNAS (U.S.A.) 84: 7280-84 (1987); Chaterjee, et al., J. Biol.Chem., 261:9929-37 (1986). The beta chains have also been chemicallycrosslinked. Kavanaugh, et al., Biochemistry, 27: 1804-8( 1988).Kavanaugh notes that the beta N-termini are 16 Å apart in the T stateand 20 Å apart in the R state. Not surprisingly, the introduction of aDIDS bridge between the N- termini of T state hemoglobin hindered theshift to the R state, thereby decreasing the O₂ affinity of themolecule. While the Kavanaugh analogue has desirable oxygen binding andrenal clearance characteristics, it too is obtained in low yield.

Hoffman and Nagai, USP 5,028,588 suggest that the T state of hemoglobinmay be stabilized by intersubunit (but intratetrameric) disulfidecrosslinks resulting from substitution of cysteine residues for otherresidues. A particularly preferred crosslink was one connecting beta GlyCys with either alpha G17 (Ala→Cys) or G18 (Ala→Cys).

F. Artificially Crosslinked Hemoglobin (Polymerizing)

Bonsen, U.S. Pat. No. 4,001,401, U.S. Pat. No. 4,001,200, and U.S. Pat.No. 4,053,590 all relate to polymerization of red blood cell-derivedhemoglobin by chemical crosslinking. The crosslinking is achieved withthe aid of bifunctional or polyfunctional crosslinking agents,especially those reactive with exposed amino groups of the globinchains. The result of the crosslinking reaction is a polydispersecomposition of covalently cross-linked aggregates.

Bonhard, U.S. Pat. No. 4,336,248 discloses chemical crosslinking ofhemoglobin molecules to each other, or to serum proteins such asalbumin.

Bonhard, U.S. Pat. No. 4,777,244 sought to stabilize thedialdehyde-cross-linked hemoglobins of the prior art, which tended topolymerize further while in storage, by adding a reducing agent tostabilize the azomethine bond.

Bucci, U.S. Pat. No. 4,584,130, at col. 2, comments that "thepolyhemoglobin reaction products are a heterogeneous mixture of variousmolecular species which differ in size and shape. The molecular weightsthereof range from 64,500 to 600,000 Daltons. The separation ofindividual molecular species from the heterogeneous mixture is virtuallyimpossible. In addition, although longer retention times in vivo areobtained using polyhemoglobins, the oxygen affinity thereof is higherthan that of stroma-free hemoglobin."

According to Tye, U.S. Pat. No. 4,529,179, "most workers have chosen toform the random intermolecular crosslinked polymers of hemoglobinbecause they believed that the 65,000" Dalton tetramer was filtered bythe glomerulus . . . . Usually the amino groups of lysine on the surfaceof the hemoglobin molecule are coupled with a bifunctional reactant suchas glutaraldehyde or suberimidate. There are 42 lysines available forreaction per hemoglobin tetramer so that one can get an infinite numberof different inter or! intra molecular crosslinks making variouspolymers of hemoglobin . . . . The random polymerization is difficult tocontrol and gives a range between two and ten tetramers per polymer . .. . No one has yet standardized an analytical scheme to establish lot tolot variability of structure and function . . . . Polymerizedpyridoxylated hemoglobin has! a profound chemical heterogeneity makingit difficult to study as a pharmaceutical agent."

G. Fused Genes and Proteins, Generally

Genes may be fused together by removing the stop codon of the firstgene, and joining it in phase to the second gene. Parts of genes mayalso be fused, and spacer DNAs which maintain phase may be interposedbetween the fused sequences. The product of a fused gene is a singlepolypeptide, not a plurality of polypeptides as is expressed by apolycistronic operon. Different genes have been fused together for avariety of purposes. Thus, Gilbert, U.S. Pat. No. 4,338,397 inserted arat preproinsulin gene behind a fragment of the E. coli penicillinasegene. His purpose was to direct E. coli transformants to secrete theexpression product of the fused gene. Fused genes have also beenprepared so that a non- antigenic polypeptide may be expressed alreadyconjugated to an immunogenic carrier protein.

The use of linker DNA sequences to join two different DNA sequences isknown. These linkers are used to provide restriction sites for DNAcleavage, or to encode peptides having a unique character thatfacilitates purification of the encoded fusion protein or a fragmentthereof. See, e.g., Rutter, U.S. Pat. No. 4,769,326.

Hallewell, et al., J. Biol. Chem., 264: 5260-68 (1989) prepared ananalogue of CuZn superoxide dismutase. Each dismutase molecule is adimer of two identical subunits; a copper ion and a zinc ion areliganded to the subunit. The dimer interaction in CuZn superoxidedismutase is so strong that the subunits have not been separated withoutinactivating the enzyme. The enzyme has considerable conformationalsimilarity to immunoglobulins; Hallewell, et al., joined two humansuperoxide dismutase genes, either directly or with DNA encoding a19-residue human immunologlobulin IgA1 hinge region and expressed thefused genes in a transformed host. In attempting to express the directlyjoined genes, recombination occurred to eliminate one of the tandemgenes in some plasmid molecules. Hallewell, et al., postulated that thedirect connection distorted the dimer, causing the exposure ofhydrophobic areas which then had a toxic effect. This would haveprovided selection pressure favoring gene deletion. No recombination wasdetected with the IgA1 linker construction.

Hoffman, et al., WO88/09179 describe the production, in bacteria andyeast, of hemoglobin and analogues thereof. The disclosed analoguesincluding hemoglobin proteins in which one of the component polypeptidechains consists of two alpha or two beta globin amino acid sequencescovalently connected by peptide bonds, preferably through anintermediate linker of one or more amino acids, without branching. Innormal hemoglobin, the alpha and beta globin subunits are non-covalentlybound.

SUMMARY OF THE INVENTION

The present invention relates to multimeric hemoglobin-like proteinswherein two or more tetramers or pseudotetramers are covalently bonded.Between any pair of covalently linked tetramers, the covalent linkagemay take the form of a crosslink between two cysteine residues ofdifferent polypeptide chains, or of a peptide linker connecting the"carboxy most" residue of a globin-like domain of one tetramer with the"amino most" residue of a similar domain of a second tetramer.

Preferably, the multimeric hemoglobin-like protein-containingcomposition is at least 50% monodisperse, more preferably, at least 95%raonodisperse.

Although free hemoglobin purified from natural sources may bepolymerized by chemical crosslinking to increase halflife via increasedmolecular weight, and to reduce oncotic pressure, all such preparationsare heterogeneous. Monodispersability can be achieved only by laboriouspurification.

The present invention provides means of exerting strict control over thedegree of polynerization of hemoglobin tetramers. The ability tostrictly control formation of multimers will greatly facilitatepurification and characterization of the final product and will reducethe chance of adverse reaction to minor components. It is also believedthat a more monodisperse composition will have greater consistency ofclinical effect.

Hemoglobin also may be made by expression of alpha and beta globin genesin the same or different host cells, and subsequent assembly of theexpressed alpha and beta globins, with heme, to form hemoglobin. Whilethe introduction of suitable Cys codon mutations into the globin genesfacilitates the production of a crosslinked multimeric hemoglobin, theexpression product in general, will not be essentially monodisperse.Hemoglobin is composed of two alpha and two beta globin subunits. Bothalpha globin subunits are natively expressed from a single alpha globingene, and both beta globin subunits, from a single beta globin gene.Thus, if an alpha globin gene is expressed which contains a single Cyscodon substitution, the assembled tetramer will contain two alpha globinsubunits, each with a crosslinkable Cys. One Cys could crosslink to asecond tetramer, and the other to a third, thus resulting in formationof a higher order oligomer.

In one embodiment, the multimeric protein is an octamer consistingessentially of two tetramers which are covalently crosslinked. To avoidunwanted polymerization, each tetramer has only a single participatingcysteinyl residue, whose thiol groups are reacted either with each other(under oxidizing conditions, forming a disulfide bond) or with athiol-reactive crosslinking agent, to form the crosslink.

A fused gene which encodes a single polypeptide comprising twoglobin-like domains may be mutated so as to provide an externallycrosslinkable Cys in only one of the two otherwise substantiallyidentical domains of the resulting pseudodimeric polypeptides. Thispseudodimer may then be assembled with the complementary subunits toform a tetramer with only the single cysteine. Two such tetramers,finally, may be crosslinked to obtain the octamer, preferably inessentially monodisperse form.

If the formation of a higher order multimer, such as a dodecamer, isdesired, the component pseudotetramers, each having a single externallycrosslinkable cysteine, are each covalently attached to a reactive siteof a polyfunctional crosslinker having a suitable half-life in thebloodstream.

Another way of obtaining a multimeric hemoglobin instead of crosslinkingtwo or more pseudotetramers, is to combine their pseudodimeric subunitsinto a single pseudooligomer that is shared by all of the componentpseudotetramers of the multimeric hemoglobin. For example apseudooctameric polypeptide, comprising eight alpha globin-like domains,joined covalently by peptide bonds (typically with a peptide spacer),may be assembled with eight individual beta globin-like subunits to forma tetra-tetrameric human hemoglobin-like protein. Higher order multimersmay be prepared simply by expressing a suitable pseudooligomer andassembling it with the complementary monomeric subunits.

The preparation of multimeric hemoglobins with a genetically fusedpseudooligomeric backbone avoids the disadvantages of chemicalcrosslinking. The latter is inefficient and often requires deoxygenationof the hemoglobin solution and the presence of another molecule (e.g.,inositol hexaphosphate or 2,3-DPG) to prevent competing reactions.

In the embodiments discussed above, an essentially monodispersemultimeric hemoglobin is achieved by limiting the number of externallycrosslinkable cysteines to one per tetramer. However, it is possible tohave more than one externally crosslinkable cysteine per tetramer,provided that they are so positioned that after one is crosslinked to aforeign tetramer, other foreign tetramers are sterically prevented fromcrosslinking to the remaining cysteines of the original tetramer.

The multimeric proteins of the present invention, particularly at higherlevels of polymerization, may prolong the half-life of recombinanthemoglobin by reducing extravasation and glomerular filtration ofdissociated subunits in vivo compared to native human hemoglobin.Studies of halflife as a function of macromolecular size indicate acorrelation between increased size and increased circulatory halflifefor chemically crosslinked Hb as well as other macromolecules.Preferably, in humans, the half-life exceeds 9 hours at a dose of atleast 1 gm/kgm body weight. This would be expected to correspond to ahalf-life of about 3 hours in rats given a comparable dose.

Intravascular retention may also be enhanced by engineering the tetramercrosslinking sites so that the haptoglobin binding sites of thetetramers are wholly or partially occluded. Independent mutations mayalso be made to sterically hinder haptoglobin binding, or toelectrostatically repel or sterically hinder the approach of agentswhich otherwise might degrade the crosslink.

The multimeric proteins of the present invention may also increaseoncotic pressure because the number of oxygen binding heme groups perpolytetramer of order "n" is "n" times the number per tetramer.Independent of size, the oncotic pressure for a given concentration ofheme groups in a solution of polytetrameric Hb is expected to be (1/n)times that of an equimolar solution of heme contained in tetrameric Hb.Because of oncotic pressure effects, the maximum concentration of freetetrameric Hb that may be introduced into the blood stream is less on aper volume basis than the concentration of Hb normally carried in intactred blood cells. Reduction of oncotic pressure is therefore useful inincreasing the per volume oxygen carrying capacity of a bloodsubstitute.

In a preferred embodiment, one or more globin-like domains containmutations which reduce the oxygen-binding affinity of the hemoglobinanalogue in solution so as to approach the oxygen-bindingcharacteristics of whole blood.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Plasmid pSGE1.1E4. This plasmid bears a poly-cistronic operonwhich comprises the pTAC promoter and genes encoding a di-alpha globinand a beta globin. It also carries tetracycline and ampicillinresistance genes, and the lacI gene.

FIGS. 2a-2e Show the sequence SEQ ID NO:1! of a preferred synthetic genefor expression of (des-Val)-alpha-(Gly)-alpha and des-Val beta globin.This gene is carried by pSGE1.1E4. A show the region (EcoRI to PstI)containing Shine-Delgarno ribosomal binding sites (SD#1 and SD#2), thesequence expressing the octapeptide (Met . . . Glu) (SEQ ID NO:25) whichserves as a cotranslational coupler, and the sequence encoding the twonearly identical alpha globin-like polypeptides and the interposedGly-Gly linker. The first alpha globin sequence begins "Met-Leu", thatis, it contains an artifactual methionine, omits the valine which is thenormal first residue of mature alpha globin, and continues with thesecond residue, leucine. The residues are numbered 1 to 141 (SEQ IDNO:26). The second alpha globin sequence begins Val-Leu, immediatelyafter the underlined "Gly-Gly" linker. The residues are numbered 1' to141' (SEQ ID NO:27). Start and stop codons are underlined. B show theanalogous region (PstI to HindIII) containing the coding sequence fordes-Val beta globin.

The beta residues are numbered 1 to 146 (SEQ ID NO:28). A and B areconnected at the PstI site to form a single polycistronic operon.

When a three letter amino acid code is singly underlined, this indicatesthat the residue is a potential site for an Xaa →Cys mutation to providea crosslinkable site. The mutations should be made asymmetrically, i.e.,in only one region of a di-alpha or di-beta gene, so only one crosslinkis added per tetramer. While, in FIGS. 2a-2e, the sites are marked onlyon the first copy of the alpha gene, they could instead be in the secondcopy. For convenience, the appropriate beta globin mutation sites arealso marked. However, these mutations should be made in only onebeta-globin of a di-beta globin gene.

Doubly underlined amino acid codes identify sites where formation of twodisulfide bonds (or per subunit) would be sterically hindered, so use ofa di-alpha or di-beta construction is unnecessary.

Residues which are candidate sites for mutations to block haptoglobinbinding are boxed.

FIG. 3 is a stylized representation of one form of pseudooctameric Hgb,in which the octameric hemoglobin is formed by linking or crosslinkingtwo molecules of an asymmetric di-alpha Hgb.

FIGS. 4a-4b depict coiled coil crosslinkers suitable for joining (a)four or (b) six Hgb tetramers. FIG. 4c is a top view of a 4-helicalbundle, with attachment sites marked.

FIGS. 5a-5c Schematics showing how cysteine mutations can favorformation of octamer without genetic fusion of subunits.

FIG. 6 Proposed alpha₁ -beta₂ globin pseudodimer.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Definitions

A hemoglobin is a protein which contains heme (ferroprotoporphyrin IX)and that binds oxygen at a respiratory surface (skin, gills, trachea,lung, etc.) and transports the oxygen to inner tissues, where it isreleased and used for metabolism. In nature, low molecular weighthemoglobins (16-120 kilodaltons) tend to be enclosed in circulating redblood cells while the larger polymeric hemoglobins circulate freely inthe blood or hemolymph.

For the purpose of the appended claims, a hemoglobin-like protein is aprotein with the following characteristics:

(a) it is sufficiently soluble in blood to be clinically useful as ablood substitute;

(b) it reversibly binds oxygen, under physiological conditions;

(c) each polypeptide chain comprises at least one globin-like domain (asdefined below); and

(d) each globin-like domain bears (or is capable of incorporating) aheme prosthetic group;

A multimeric hemoglobin-like protein is further characterized asfollows:

(e) it is composed of two or more polypeptide chains;

(f) it is composed of two or more tetramers, each tetramer comprisingfour globin-like domains, and

(g) each component tetramer is covalently attached to at least one othercomponent tetramer.

Preferably, the hemoglobin-like proteins of the present invention have aP₅₀ of 2 to 45 torr, more preferably 24 to 32 torr, at 37° C., in blood.Preferably, they also exhibit some degree of cooperativity. Also, theydesirably have an intravascular retention at least comparable to that ofnormal human hemoglobin administered as a blood substitute.

Tetrameric hemoglobin-like proteins have four globin-like domains,octameric hemoglobin-like proteins have eight globin-like domains, andso forth. The term "multimeric" covers any hemoglobin-like proteincomprising (4×n) globin-like domains, where n>l.

A pseudomeric hemoglobin-like protein is one for which the number ofglobin-like domains is greater than the number of component polypeptidechains, i.e., at least one chain comprises at least two globin-likedomains. The pseudoheterotetrameric hemoglobin-like proteins, forexample, may be composed of (a) one di-alpha globin-like and two betaglobin-like polypeptides, (b) two alpha globin-like and one di-betaglobin-like polypeptides, (c) one di-alpha globin-like and one di-betaglobin-like polypeptides, (d) one fused alpha/beta globin-likepolypeptide and separate alpha and beta globin-like polypeptides, or (e)two fused alpha/beta globin-like polypeptides. The term "tetramer"includes "pseudotetramers."

A "genetically fused hemoglobin" is a hemoglobin-like protein comprisingat least one "genetically fused globin-like polypeptide", (globinpseudooligomer), the latter comprising two or more globin-like domainswhich may be the same or different and which are connected directly, orthrough an amino acid or peptide linker. A di-alpha globin-likepolypeptide is one which consists essentially of two alpha-globin-likepolypeptide sequences (domains) connected by peptide bonds between thenormal C- terminus of the first alpha-globin-like polypeptide (domain)and the normal N-terminus of the second alpha-globin-like polypeptide(domain). These two sequences may be directly connected, or connectedthrough a peptide linker of one or more amino acids; the term "peptidebonds" is intended to embrace both possibilities. Alpha globin chainscrosslinked at the N- and C-terminals other than by peptide bonds (e.g.,by DIDS) are not di-alpha globins. The di-alpha globin-like polypeptidemust be capable of folding together with beta globin and incorporatingheme to form functional hemoglobin-like protein. The di-beta globin-likepolypeptide is analogously defined. A di-alpha or di-beta globin-likepolypeptide with a mutation in only one of the component domains iscalled "asymmetric".

It is also possible to provide an "alpha/beta-globin-like pseudodimer"in which an alpha globin-like sequence is connected by peptide bonds toa beta globin-like sequence. This "alpha/beta globin-like polypeptide",and the di-alpha and di-beta globin-like polypeptides, may collectivelybe referred to as "pseudodimeric globin-like polypeptides"or as"diglobins". By extension, a hemoglobin-like protein comprising adi-alpha, a di-beta, or a alpha/beta globin-like polypeptide is a"pseudotetramer".

Pseudotetramers which bear only a single externally crosslinkablecysteine may be referred, by way of shorthand, as "mono-cys" molecules.However, the use of this term should not be taken as implying that thetetramer may not comprise other cysteines. A "mono-cys" pseudotetrameris merely one which has only a single cysteine which can participate toa significant degree in crosslinking reactions with a cysteine residueof a second pseudotetramer.

A hemoglobin-like protein is said to be heteromeric if at least two ofits globin-like domains are different. Since conventional humanhemoglobin is composed of two alpha globins and two beta globins, it isa heterotetramer. A multimeric human hemoglobin-like protein is aheteromer wherein each tetramer or pseudotetramer has two human alphaglobin-like domains and two human beta globin-like domains.

The Globin-Like Domain

The globin-like domains may be, but need not be, one per polypeptidechain, and they need not correspond exactly in sequence to the alpha andbeta globins of normal human hemoglobin. Rather, mutations may beintroduced to alter the oxygen affinity (or its cooperativity, or itsdependence on pH, salt, temperature, or other environmental parameters)or stability (to heat, acid, alkali, or other denaturing agents) of thehemoglobin, to facilitate genetic fusion or crosslinking, or to increasethe ease of expression and assembly of the individual chains. Guidanceas to certain types of mutations is provided, e.g., by Hoffman andNagai, U.S. Pat. No. 5,028,588, and Ser. No. 07/443,950, incorporated byreference herein. The present invention further includes molecules whichdepart from those taught herein by gratuitous mutations which do notsubstantially affect biological activity.

A "globin-like domain" is a polypeptide domain which is substantiallyhomologous with a globin subunit of a naturally occurring hemoglobin. A"vertebrate," "mammalian" or "human" globin-like domain is one which issubstantially homologous with a globin subunit of, respectively, anaturally occurring vertebrate, mammalian or human hemoglobin.

A human alpha globin-like domain or polypeptide is native human alphaglobin or a mutant thereof differing from the native sequence by one ormore substitutions, deletions or insertions, while remainingsubstantially homologous (as hereafter defined) with human alpha globin,and still capable of incorporating heme and associating with betaglobin. The term "human alpha globin-like domain" is intended to includebut not be limited to naturally occurring human alpha globins, includingnormal human alpha globin. A beta globin-like domain or polypeptide isanalogously defined. Subunits of animal hemoglobins or mutants thereofwhich are sufficiently homologous with human alpha or beta globin areembraced by the term "human alpha or beta globin-like domain orpolypeptide." For example, the subunits of bovine hemoglobin are withinthe scope of these terms.

In determining whether a polypeptide is substantially homologous toalpha (or beta) globin, sequence similarity is an important but notexclusive criterion. Sequence similarity may be determined byconventional algorithms, which typically allow introduction of a smallnumber of gaps in order to achieve the best fit. A humanalpha-globin-like domain will typically have at least about 75% sequenceidentity with wild-type human alpha globin, and greater homology withhuman alpha globin than with human beta globin. However, a polypeptideof lesser sequence identity may still be considered "substantiallyhomologous" with alpha globin if it has a greater sequence identity thanwould be expected from chance and also has the characteristic higherstructure (e.g., the "myoglobin fold") of alpha globin, the ability toincorporate heme, and oxygen-binding activity. (Note that, as elsewhereexplained, an alteration in oxygen affinity (P50), intravascularretention, or cooperativity may be desired, and does not render themutant nonhomologous if it can still contribute to reversibleoxygen-binding activity.) By way of comparison, Artemia's heme-bindingdomains are considered homologous with myoglobin even though the primarysequence similarity is no more than 27%, as alignment of the heme-binding domains around their conserved residues and. the residuesconserved in other hemoglobins (i.e., involved in heme contacts or indetermining the relationship of the helical segments to each other)suggested that the Artemia domains possessed the classical globinhelices A to H with their corresponding turns, as well as variousconserved globin family residues. Also, among the serine proteaseinhibitors, there are families of proteins recognized to be homologousin which there are pairs of members with as little as 30% sequencehomology.

Over a hundred mutants of human hemoglobin are known, affecting both thealpha and beta chains, and the effect of many of these mutations onoxygen-binding and other characteristics of hemoglobin are known. Thehuman alpha and beta globins themselves differ at 84 positions. Inaddition, interspecies variations in globin sequence have beenextensively studied. Dickerson, Hemoglobin: Structure, Function,Evolution and Pathology, ch. 3 (1983) reported that in 1982, the 60known vertebrate alpha globins had identical residues at 23 of their 141positions, while for the 66 vertebrate beta globins considered, 20 ofthe 146 amino acids are identical. The 60 vertebrate myoglobins, whichalso belong to the globin family, had 27 invariant amino acids out of153 positions. If only mammals are considered, then the invariant aminoacids are 50/141 for the alpha globins, 51/146 for the beta globins, and71/153 for the myoglobins. Invariant positions cluster around thecenters of activity of the molecule: the heine crevice and theintersubunit contacts. Of the variable amino acids, some diverge fromthe consensus sequence for only a small fraction of the speciesconsidered.

The number of total differences between human alpha globin and selectedother vertebrate alpha globins is as follows: rhesus monkey (4), cow(17), platypus (39), chicken (35), human zeta (embryonic) (61), carp(71), and shark (88). For invertebrate globins the divergences are sealamprey (113), mollusc (124), Glycera (marine bloodworm) (124) andChironomus (midge) (131). Turning to the beta globin family, thedifferences of human beta globin from other vertebrate beta globins arerhesus monkey (8), human delta globin (10), cow beta globin (25), cowgamma globin (33), human gamma globin (39), human epsilon (embryonic)globin (36), platypus (34), chicken (45), shark (96), sea lamprey (123),mollusc (127), Glycera (125) and Chironomus (128).

Many of these differences may be misleading--variable amino acids mayexhibit only "conservative substitutions⃡ of one amino acid for another,functionally equivalent one. A "conservative substitution" is asubstitution which does not abolish the ability of a globin- likepolypeptide (or domain) to incorporate heme and to associate with alphaand beta globin subunits to form a tetrameric (or pseudotetrameric)hemoglobin-like protein which, in keeping with the definition thereof,will reversibly bind oxygen. The following resources may be used toidentify conservative substitutions (and deletions or insertions):

(a) data on functional hemoglobin mutants (over a hundred such mutantsexist);

(b) data on sequence variations among vertebrate, especially mammalian,alpha globins and beta globins;

(c) data on sequence variations among vertebrate, especially mammalian,myoglobins;

(d) data on sequence variations between vertebrate and invertebrateglobins, or among the invertebrate globins;

(e) data on the three-dimensional structures of human hemoglobin andother oxygen-binding proteins, and molecular modelling software forpredicting the effect of sequence changes on such structures; and

(f) data on the frequencies of amino acid changes between members offamilies of homologous proteins (not limited to the globin family). See,e.g., Table 1-2 of Schulz and Schirmer, Principles of Protein Structure(Springer-Verlag: 1979) and FIGS. 3-9 of Creighton, Proteins: Structureand Molecular Properties (W. H. Freeman: 1983).

While the data from (a)-(d) is most useful in determining tolerablemutations at the site of variation in the cognate proteins, it may alsobe helpful in identifying tolerable mutations at analogous siteselsewhere in the molecule. Based on the data in category (f), thefollowing exchange groups may be identified, within which substitutionsof amino acids are frequently conservative:

I small aliphatic, nonpolar or slightly polar residues--Ala, Ser, Thr(Pro, Gly)

II negatively charged residues and their amides--Ash, Asp, Glu, Gln

III positively charged residues--His, Arg, Lys

IV large aliphatic nonpolar residues--Met, Leu, Ile, Val (Cys)

V large aromatic residues--Phe, Tyr, Trp

Three residues are parenthesized because of their special roles inprotein architecture. Gly is the only residue without a side chain andtherefore imparts flexibility to the chain. Pro has an unusual geometrywhich tightly constrains the chain. Cys can participate in di-sulfidebonds which hold proteins into a particular folding. Note that Schulzand Schimer would merge I and II above. Note also that Tyr, because ofits hydrogen bonding potential, has some kinship with Ser, Thr, etc.

In general, functionality is less likely to be. affected by mutations atsurface residues, at least those not involved in either the heme creviceor the subunit contacts. in addition, "loops" connecting alpha helices,as well as free amino or carboxy termini, are more tolerant of deletionsand insertions.

A "Met FX alpha globin" is an alpha globin-like polypeptide comprisingan N-terminal methionine, a oligo-peptide which acts as a recognitionsite for Factor Xa (e.g., Ile-Glu-Gly-Arg)(SEQ ID NO:2), and an alphaglobin-like sequence (e.g., Val-His-Leu-Thr-Pro . . . ) (SEQ ID NO:3)which may correspond to wild-type alpha globin or to a mutant thereof astaught herein. The term "Met FX alpha globin" is some-times abbreviatedas "FX alpha globin". "FX beta globin" is an analogously defined betaglobin-like polypeptide.

"Met-alpha globin" is an alpha globin-like polypeptide with an extraN-terminal methionine. The second amino acid is valine, which is thefirst amino acid of mature wild-type alpha globin. Met-beta globin isanalogously defined. A "Des-FX alpha globin" gene (or "dFX alphaglobin") is a Met-alpha globin gene obtained by excising the FX codonsfrom a Met-FX alpha globin gene. Note that "Met-Hgb" is used to refer tomethionyl Hgb formed from methionyl-alpha globin and methionyl-betaglobin.

"Des-Val-alpha globin" (or "dVal alpha globin") is an alpha globin-likepolypeptide wherein methionine is substituted for the valine whichbegins the sequence of mature wild-type alpha globin. Des-Val-betaglobin is analogously defined. Des-Val-alpha/alpha globin(di-Des-Val-alpha globin) is a "di-alpha globin" in which a"Des-Val-alpha" sequence is linked via an appropriate peptidyl linker toan alpha globin-like sequence which begins with Val.

Low Affinity Mutants

The term "low affinity hemoglobin-like protein" refers to ahemoglobin-like protein having a P₅₀ which is at least 10% greater thanthe P₅₀ of cell free normal hemoglobin A_(o) under the same conditions.Preferably, the protein, if used as a blood substitute, qualifies as alow affinity protein, and more preferably, its P₅₀ is closer to the P₅₀of whole blood cells than to that of cell free hemoglobin.

Low affinity mutant hemoglobins, i.e., those with "right shifted" oxygenequilibrium binding curves relative to cell-free normal hemoglobin, havemany potential uses. Most notably, mutant hemoglobins that have anoxygen affinity similar to whole red blood cells may be used as anoxygen- carrying transfusion substitute in place of donated red bloodcells, eliminating the risk of infection and alleviating problems withsupply. Cell-free native human hemoglobin cannot function as atransfusion substitute, among other reasons because oxygen is bound tootightly. In addition, because cell-free hemoglobin solutions do not needto be cross-matched and are expected to have a longer shelf life thanwhole blood, low affinity hemoglobin solutions may be widely used insituations where whole blood transfusion is not feasible, for example inan ambulance or on a battlefield. Mutant hemoglobins that have an evenlower oxygen affinity than red blood cells may in fact delivery oxygenmore effectively in many situations. Mutant hemoglobins that have asomewhat higher oxygen affinity than whole blood (but a lower affinitythan cell-free native human hemoglobin) will still function as anadequate transfusion substitute and may in fact deliver oxygen moreeffectively than red blood cells in some situations. This is becauseoxygen is released directly to plasma form hemoglobin-based solutions,without the need to diffuse through the red cell membrane, and becausecell-free hemoglobin may penetrate into regions not accessible to redblood cells. As an example, low affinity mutant hemoglobin is expectedto deliver oxygen effectively during coronary artery balloon angioplastyprocedures, whereas circulation of red blood cells is obstructed duringsuch procedures. Low affinity mutant hemoglobin may also be useful as aperfusion component in organ preservation prior to transplantation or asa mammalian cell culture additive.

Possible low affinity mutants are discussed in detail, by way of exampleand not of limitation, in Table 1 (natural low affinity hemoglobinmutants) and Table 2 (candidate non-naturally occurring low affinityhemoglobin mutants) of Hoffman, et al., U.S. Pat. No. 5,028,588. Lowaffinity mutants of particular interest are the Presbyterian (betaLys¹⁰⁸) beta Phe⁶³, beta Ile⁶⁷, and Kansas (beta Thr¹⁰²) mutants.

An unexpected and surprising change in oxygen binding characteristics ofhemoglobin was observed upon replacement of the N-terminal valine withmethionine. Hemoglobin A_(o) purified from blood has a P₅₀ value of 4.03with N=2.8 when measured at 25° C. DesFX-Hgb produced in E. coli, ahemoglobin identical to A_(o) except for the addition of a methionine atthe N-termini of the alpha and beta chains, has essentially the same P₅₀and N values. Thus, the addition of a methionine, without altering theadjacent valine residue, has little or no effect on oxygen binding. Onthe other hand, a higher P₅₀ value, 6.6, was observed for desVal-Hgbproduced in E. coli, a hemoglobin in which the normal N-terminal valineof each chain was replaced with methionine. Cooperativity, as measuredby N, was virtually the same, however, for all three molecules.

A similar comparison was made for two hemoglobins each containing thePresbyterian mutation, one produced in E. coli and one in yeast. The E.coli hemoglobin was constructed with a Des-Val alpha chain, i.e., theN-terminus had the normal valine replaced with methionine. Oxygenbinding was characterized by P₅₀ =19.8, N=2.5 at 25° C. and by P₅₀ =34.5and N=2.5 at 37° C. The corresponding yeast coding region begins with anadditional methionine codon in front of the normal valine codon. Becausethis initial methionine is removed post translationally in vivo, thepurified hemoglobin has a normal N-terminal valine. For this molecule,P₅₀ =23 to 25 and N=2.5 when measured at 37° C. Thus, in the aboveinstances, the replacement of an N-terminal valine with an N- terminalmethionine increased the P₅₀ value. Under physiological conditions, itis expected that the genetically fused Presbyterian hemoglobin producedin E. coli will deliver 20-30% more oxygen than the similar hemoglobin,with its altered N-terminus, produced in yeast.

High Affinity Mutants

The term "high affinity hemoglobin-like protein" refers to ahemoglobin-like protein having a P₅₀ which is at least 10% less than theP₅₀ of cell free hemoglobin A_(o) under the same conditions.

High affinity mutant hemoglobin may have utility in certain situations.For example, perfluorocarbon-based blood substitute preparations areunder clinical study for enhancement of radiation therapy and certainchemotherapy treatments of solid tumors (Dowling, S., Fischer, J. J.,and Rockwell, S. (1991) Biomat. Art. Cells Immobil. Biotech, 19, 377;Herman, T. S. and Teicher, B. A. (1991) Biomat. Art. Cells and Immobil.Biotech, 19, 395; Holden, S. A., Teicher, B. A. and Herman, T. S. (1991)Biomat. Art. Cells and Immobil. Biotech, 19, 399.) The basis of theseinvestigations is the fact that oxygen is a required component of thecell toxicity action of radiation and certain chemotherapy reagents.Solid tumors frequently exhibit extremely low partial oxygen pressure inthe interior of the tumor, rendering therapy inefficient.Perfluorocarbon-based oxygen-carrying solutions appear to dramaticallyenhance certain tumor therapies, and hemoglobin- based blood substitutesare expected to have a similar utility. It is likely that cell-freehemoglobin unlike whole red blood cells, will be able to penetrate theinterior region of tumors for delivery of oxygen. Actual percent ofoxygen released by a cell-free hemoglobin preparation is not a directfunction of P₅₀ but rather depends on the shape of the oxygenequilibrium binding curve between the two pressures representing thepartial oxygen pressure of the lungs (where oxygen is loaded ontohemoglobin) and the partial pressure of the tissue where oxygen isunloaded. Therefore, it is possible that a high affinity mutanthemoglobin would be preferred as a tumor therapy adjuvant. A highaffinity hemoglobin would retain its bound oxygen throughout the normalcirculatory system, where partial oxygen pressure remains relativelyhigh, but release its oxygen in the extremely oxygen-depleted tumorinterior. Normal or low affinity hemoglobin might have less oxygenavailable for release by the time it reaches the interior of the tumor.

Naturally occurring high affinity hemoglobin mutants are also known, seeBunn and Forget, Table 14-1, and candidate non-naturally occurring highaffinity hemoglobin mutants may be proposed in view of the known mutantsand hemoglobin structure. Particularly preferred high affinity mutantsare set forth in Table 400.

It should be noted that genetic fusion and crosslinking can affectoxygen binding affinity.

Cysteine Mutations and Disulfide Bridge Formation

Cysteine mutations are of value for increasing the stability of thetetramer (See U.S. Pat. No. 5,028,588 and Ser. No. 07/443,950, nowabandoned. They also facilitate constructing poly(tetrameric) (n>=2)hemoglobins. This is because the cysteines on adjacent tetramers(including pseudotetramers) can be oxidized to form a disulfide bridge,covalently coupling the tetramers. In addition, the thiol groups ofcysteines may be reacted with a variety of crosslinking agents.

A variety of sites are available for introduction of cysteines into ahemoglobin-like protein.

The criteria governing site selection are: (1) the mutation does notaffect functionality; (2) the side chain is accessible to water in oxyor deoxy structure; (3) the site should lie on the surface of the foldedprotein; (4) the sulfhydryl of the side chain should extend away fromthe surface rather than toward the interior of the molecule; (5) thesite should be in a portion of the molecule that is not directlyinvolved in the R->T transition; (6) the change should be in a portionof the molecule that does not have a tightly fixed position (suchregions generally give indistinct X-ray diffraction patterns); (7) themutations will not destroy the local secondary structure, i.e., avoidpro->cys mutations, which might result in a refolding problem; and (8)if possible, a conservative change should be made such as ser->cys orala>cys. A mutation does not necessarily have to meet all of the aboverequirements to be useful. For example, one might envision a site thatis involved in the R->T transition (cf. 5 above) but confers abeneficial change in P₅₀ (cf. 1 above) because of that involvement. Themost important considerations are that the mutation does not abolish O₂binding, before or after crosslink formations, and that the cysteine isaccessible for participation in the desired crosslinking reaction.

Candidate sites on the alpha surface include: his72, asn 78, asn68,ala71, thr67, lys7, lys11, thr8, ala12, thr118, lys16, ala45, glu116,gly15, his112, thr24, glu23, lys60, lys56, his50, gly51, glu53, ser49,asp47, gln54, his45, lys90, ala82, lys61, ala19, his20, asp85, ser81,asp75, asp74, lys139, asp64, and gly18 (total 40 amino acids).

Candidate sites on the beta surfaces includes: asp79, his2, leu3, thr4,glu6, ser9, thr12, ala13, gly16, lys17, val18, asn19, val20, asp21,glu22, lys65, ser72, ala76, his77, asp79, asn80, gly83, ala86, thr87,glu90, lys95, lys59, glu43, ser44, asp47, ser49, thr50, ala53, asp52,lys61, glu121, lys120, thr123, lys66, asp73, ala62, his116, his117,(total 45 amino acids).

There are a number of naturally occurring mutants which already showmutations at these sites. These are listed below:

    ______________________________________                                        Residues   Region          Mutation                                           ______________________________________                                        19         AB1             ALA->GLU                                                                      ALA->ASP                                           54         E3              GLN->ARG                                                                      GLN->GLU                                           71         E20             ALA->GLU                                           75         EF4             ASP->GLY                                                                      ASP->HIS                                                                      ASP->TYR                                                                      ASP->ASN                                           81         F2              SER->CYS                                           47         CE5             ASP->GLY                                                                      ASP->HIS                                                                      ASP->ASN                                           ______________________________________                                    

If the pseudo-octamer (n=2) is formed by directly linking twopseudo-tetramers via a disulfide bond, the halflife in serum may beinfluenced by the rate at which endogenous serum small molecule thiols(such as glutathione) reduce the disulfide bond. The mechanism of thesereactions involves the thiolate anion as the actual reducing species(Creighton, T. E. (1978) Prog. Biophys. Molec. Biol., 33:259-260;Creighton, T. E. (1975) J. Mol. Biol., 96:767; Creighton, T. E. (1977)J. Mol. Biol., 3:313). Thus the rate of reduction will be a function ofthe molecular electrostatic environment in the vicinity of the disulfidebond. A slower rate of reduction would be predicted if the disulfide waslocated an electrostatically negative environment, due to the repulsionof the thiolate anion. In the case of glutathione, even the unreactivetransient protonated species has a net negative charge and would berepulsed, thus further reducing the rate of disulfide reduction.

A surface or near-surface amino acid residue of di-alpha or di-betahemoglobin that is located in close proximity to a negatively chargedsurface residue might therefore be a good choice for location of asingle cysteine mutation in the di-alpha or di-beta polypeptide.Although formation of the initial disulfide bond between two suchcysteines might also be slower because of repulsion between the negativecharges on the two hemoglobin molecules in the vicinity of thecysteines, the reaction could be facilitated by use of high salt or highpH during the in vitro bond formation reaction. If carried out underdeoxy conditions in a redox buffer, the reaction might also befacilitated by temperature elevation.

    ______________________________________                                        Preferred sites for cvs mutations proximal to negative charged                ______________________________________                                        residues                                                                      alpha ser49                                                                              near asp47; naturally occurring ser49 to arg                                  has normal O.sub.2 affinity                                        alpha his20                                                                              near glu23; naturally occurring his20 to tyr,                                 gln, arg have no known undesirable properties                      alpha lys16                                                                              near glul16; naturally occurring lys to glu has                               normal O.sub.2 affinity                                            alpha his50                                                                              near glu30; naturally occurring his50 to asp                                  has no known undesirable properties                                beta thr50 near asp52; naturally occurring thr50 to lys                                  has no known undesirable properties                                beta lys65 near asp21                                                         beta asn19 near asp21                                                         ______________________________________                                    

Surface or near-surface cysteine mutations in general are not expectedto have major effects on the functionality of the hemoglobinpseudotetramer. Cysteine mutations would not be expected tosignificantly destabilize alpha helices, and surface residues are notdirectly involved in the oxygen binding properties of hemoglobin. Mostsurface residues undergo considerable motion and are not tightlyconstrained. It should also be noted that because of protein breathingmotions, the cysteine side chain would not necessarily have to pointdirectly into solution to be accessible for disulfide bond formation.

In addition to the use in construction of a pseudo- octamer, there maybe additional uses of surface cysteine mutations. These include: (1)construction of multimeric hemoglobins (n>2) by use of syntheticsulfhydryl reactive peptides with more than two reactive sites; (2)surface cysteine residues could be used to attach chelates that bindradioisotopes for imaging; and (3) surface cysteines could be used toattach bio-active peptides or other therapeutic agents to increase theircirculating half-life, or target their delivery. If the attachment ofthe drug were via a disulfide, the rate of release of the peptide fromits carrier could be controlled by neighboring residues. For uses (2)and (3) , restriction to one cysteine per di-alpha or di-beta isunnecessary.

It may be desirable to eliminate the cysteine at beta 93 of normal humanhemoglobin so that it cannot participate in polymerization reactions.This cysteine may be replaced by serine, alanine or threonine, forexample. Other wild-type cysteines may also be replaced, if desired, butit is unlikely that they participate in crosslinking reactions after thetetramer is formed.

Mutations to Reduce Haptoglobin Binding

It is presently believed that haptoglobin binding plays a role in thecatabolism of hemoglobin. If so, intravascular retention of hemoglobinmight be enhanced by mutations which inhibit haptoglobin binding.Oxyhemoglobin dissociates into alpha-beta dimers, which are then boundby haptoglobin. While much of the binding energy is associated withbinding to residues which are buried in the tetramer but exposed in thedimer, it appears that there are also secondary binding sites on thesurface of the tetramer. Though the mechanism is not clear, thehaptoglobin-bound dimers are transported to Kupffer cells in the liver,where they are catabolized.

It would be most desirable to mutate sites which both are involved inhaptoglobin binding and which are suitable for attachment of anothertetramer. Candidate mutation sites are in the alpha chain of normalhuman alpha globin, residues 1, 6, 74, 82, 85, 89, 90, 93, 118, 120-127and 139-141, and in the beta chain, residues 2, 11-40 and 131-146. It isunlikely that haptoglobin binding can be blocked merely by singlesubstitution mutation of one genetically encoded amino acid to another.However, if the above residues are replaced by a cysteine, and thecysteine is crosslinked to another molecule which is significantlylarger than the usual amino acid side chain, the steric effect ismagnified considerably and haptoglobin binding may be inhibited. Ofcourse, to retain polymerization control, these mutations should be madeasymmetrically in a pseudooligomeric polypeptide so that there is onlyone crosslink per tetramer.

It is known that even covalently crosslinked hemoglobins can beprocessed by haptoglobin; this is thought to be the result of the"breathing" of the tetramer in its oxy form sufficiently to allow thehaptoglobin access to the normally buried residues of the subunitinterfaces in question. This may be prevented by tightly crosslinkingthe globin subunits so dissociation will not occur within the time spanof interest. Unlike the mutations discussed above, these mutationsshould be made in all of the indicated subunits for maximum efficiency.

    beta37->Cys and alpha92->Cys

    beta40->Cys and alpha92->Cys

    beta97->Cys and alpha41->Cys

The above mutations all lie at the alpha₁ beta₂ and beta₁ alpha₂interfaces and lock these interfaces shut so that "breathing" does notallow haptoglobin access.

"Breathing" may also be inhibited by low oxygen affinity mutations; thetetramer then spends more time in the deoxy state, which is notsusceptible to haptoglobin attack.

Pseudomeric Globin-Like Polypeptides and PseudotetramericHemoglobin-Like Proteins Useful as Intermediates in Preparation ofMultimeric Hemoglobin-Like Proteins

In the liganded form, hemoglobin readily dissociates into αβ dimerswhich are small , enough to pass through the renal glomeruli, and Hb isthereby rapidly removed from the circulatory system. Intravenousadministration of hemoglobin in amounts far less than that needed tosupport oxygen transport can result in long term kidney damage orfailure. Ackers, G. K. and Halvorson, H. R., Proc. Nat. Acad. Sci.(U.S.A) 71, 4312-16 (1974); Bunn, H. F., Jandl, J., J. Exp. Med. 129,925-34 (1969). If dissociation into dimers is prevented, there is anincrease in intravascular half life and a substantial reduction offrenal toxicity. Lee, R., Atsumi, N., Jackobs, E., Austen, W., Viahakes,G., J. Surg. Res. 47, 407-11 (1989). The hemoglobin-like proteins of thepresent invention cannot dissociate into αβ-dimers without the breakageof a peptide bond and should have the advantages of a longerintravascular half life and reduced renal toxicity.

In the crystal structures of both deoxyhemoglobin and oxyhemoglobin theN-terminal Val residue for one α subunit and the C-terminal Arg residueof the other α subunit are only between 2 and 6 Å apart, and are boundto one another through a salt bridge in deoxyhemoglobin. Fermi, G.,Perutz, M., Shaanan, B., Fourme, R., J. Mol. Biol., 175, 159-74 (1984);Shaanan, B., J. Mol. Biol. 171, 31-59 (1983). This distance could bespanned by one or two amino acids. One extra amino acid can be added tothe C-terminal Arg residue of the α subunits by trypsin catalyzedreverse hydrolysis without significantly altering the oxygen bindingproperties. Nagai, K., Enoki, Y., Tomita, S. and Teshima,. T., J. Biol.Chem., 257, 1622-25 (1982) Preferably the di-alpha linker (if one isused) consists of 1 to 3 amino acids which may be the same or different.A Mono-Gly linker is especially preferred. In designing such a linker,it is important to recognize that it is desirable to use one or moreamino acids that will flexibly connect the two subunits, transformingthem into domains of a single di-alpha globin polypeptide.

The preparation of "di-beta" mutants is also contemplated. The distancebetween the N-terminus of one beta subunit and the C-terminus of theother is 18.4 Å in the deoxy configuration and 5.2 Å in the oxy form.Preferably, the di-beta linker consists of 2 to 9, amino acids which maybe the same or different. Glycine amino acids are particularlypreferred.

The length of the (-gly-)_(n) genetically fused link between theN-terminus of one beta chain (at beta₁, 1 Val) and the C terminus of thesecond beta chain (beta₂, 146 His) in di-beta hemoglobin may rangebetween 1 and approximately 9 glycines. In the oxy and deoxy crystalstructures of human hemoglobin A_(o), the distance between these terminiis 5.22 Å and 17.93 Å respectively (from the N-terminal nitrogen to theC terminal carbon of the carboxylate). A single glycine linker, which isa little less than 4 Å in length, may come close to linking the twotermini in the oxy structure, however, it is expected that this linkerwill fall .sup.˜ 14Å short in the deoxy structure. Significantly moreperturbation of the deoxy structure vs the oxy structure might beanticipated with this linker. Some alterations in the oxygen bindingproperties may be caused by deletion of the positive and negativecharges at the two termini and their inclusion in the amide bond. Inaddition, the linker molecule itself may destabilize the oxy structureless than the deoxy structure, and thus lead to a relative increase inoxygen affinity. Likewise, two glycines inserted as linkers may alsodifferentially stabilize the oxy structure and hence relatively increasethe oxygen affinity by the same mechanism described above.

When the number of linking glycines is increased to 5, the linker shouldjust span the cleft between the beta chain termini in the deoxystructure, and, moreover, insert added steric bulk between the terminiin the oxy structure, thus leading to a relative stabilization of deoxy(or destabilization of oxy) and perhaps resulting in a concomitantdecrease in oxygen affinity. Due to the large space between the betatermini in the deoxy (but not the oxy structure), addition of glycinelinkers in the range of 6-9 may further stabilize the oxy structure and,in the same manner, further decrease oxygen affinity.

A third form of globin pseudodimer is one comprising both alpha and betaglobin domains. A possible route to fusing alpha1 to beta2 and sostabilizing hemoglobin against α₁ β₁ /α₂ β₂ dimer formation, is to fusethe alpha1 C-terminal residue to the N-terminal residue of beta2 Chelix, creating a new C-terminus at the end of the beta2 B helix. Theoriginal beta N terminus, Val1, would be fused to the original betasubunit C-terminal residue, His146, by means of an intervening newsection of protein, thus creating a continuous polypeptide chaincomprising the alpha and beta subunits of different dimers. This chainmay be described as follows: α(1-14)-Gly₃ -β(35-146)-Gly₁₋₃ -Ala₁₃Gly₁₋₃ -β(1-34)(SEQ ID NO:29); See FIG. 6.

Inspection of the structure of human deoxyhemoglobin using a moleculargraphics computer indicates the following relevant distances. Thedistance between the Alpha1 Arg141 carboxyl carbon and Beta2 Tyr35 Natoms is approximately 8.6 Angstroms. A fully extended linear triglycinepeptide measured approximately 10.1 Angstroms from the N to C terminalresidues. This suggests that three glycine residues could be employed tospan the distance between the Arg141 and Tyr35 residues with a minimumof unfavorable steric interactions and maximum conformational freedom.The distance requirements could be different in oxyhemoglobin, and ifso, the sequence of the fusion peptide could be altered to bestaccommodate the requirements of both structures.

In human deoxyhemoglobin, the distance between the Beta2 His146 carboxylcarbon and the Beta2 Val1 nitrogen atoms is approximately 25 Angstroms.A right handed 3.6 Alpha helix constructed from a linear sequence of 13Alanine residues was found to measure 22 Angstroms from N to C terminus.With the addition of one to three glycine residues at each end of thishelix (to give Gly_(n) (Ala)₁₃ Gly_(n) where n=1 to 3), (residues130-148 of SEQ ID NO:29) it could span required distance and havesufficient conformational flexibility to avoid serious tertiary packingconflicts. Additionally, the amino acid sequence of the helix could bealtered to introduce favorable hydrogen bonds and salt bridges betweenthe new helix and the Beta2 helix against which it would pack in thefolded protein. Such interactions could aid stabilization of theengineered protein.

Glycine is the preferred amino acid in the linkers, since it is known tobe quite flexible, Cantor and Schimmel, Biophysical Chemistry, part 1,pp. 266-9 (1980), and also allows chains into which it is incorporatedto assume a more compact structure. However, the residues comprising thelinker are not limited to glycines; other residues may be includedinstead of or in addition to glycine, such as alanine, serine, orthreonine. Since these amino acids have a more restricted conformationalspace in a protein, they will likely result in more rigid linkingchains, and hence have a more pronounced effect on the relativestabilization/destabilization of the oxy/deoxy structures.

It should be understood that the minimum and maximum number of aminoacids in the linker is a function of the distance to be spanned in boththe oxy or deoxy forms, the amino acids chosen, and the propensity ofthe particular amino acid sequence to form a secondary structure. Whilea random coil is usually preferred, it is not required, and a linkerwith a large number of amino acids in a secondary structure may have thesame span as a random coil linker with fewer amino acids. A linker maycomprise, e.g., 1-3 glycines, followed by a sequence having a secondarystructure, followed by 1-3 more glycines. The translation per residue,in angstroms is 1.9 for polyproline I, 3.12 for polyproline II, 3.1 forpolyglycine II, 3.4 for an antiparallel β sheet, 3.2 for a parallelβ-sheet, 1.5 for a right handed a-helix, 2.0 for a 310 helix, and 1.15for a π helix. In a fully extended chain, the maximum translation perresidue is 3.63 Å if the repeating units are staggered and 3.8 Å if thepeptide bond is trans.

The number of amino acids in the linker may be such that a formation ofa secondary structure, such as an alpha helix or a beta-sheet, isundesirable, as the span is reduced. Certain amino acids have a greatertendency to participate in such structures. See Chou and Fasman,Biochemistry, 13:222-245 (1974), incorporated by reference. The aminoacids are ranked in order of decreasing participation below. Thepreferred linker amino acids are boldfaced. Glycine is the most suitableamino acid for this purpose. The most preferred di-alpha linkers are Glyor Gly-Gly.

    ______________________________________                                        Alpha Helix         Beta Sheet                                                Formers             Formers                                                   ______________________________________                                        Glu (1.53)          Met (1.67)                                                Ala (1.45)          Val (1.65)                                                Leu (1.34) Hα Ile (1.60) Hβ                                        His (1.24)          Cys (1.30)                                                Met (1.20)          Tyr (1.29)                                                Gln (1.17)          Phe (1.28)                                                Val (1.14)          Gln (1.23)                                                Trp (1.14)          Leu (1.22)                                                Phe (1.12) hα Thr (1.20)                                                Lys (1.07)          Trp (1.19) hβ                                        Ile (1.00)          Ala (0.97) Iβ                                        Asp (0.98)          Arg (0.90)                                                Thr (0.82)          Gly (0.81)                                                Arp (0.79)          Asp (0.80) iβ                                        Ser (0.79)          Lys (0.74)                                                Cys (0.77) iα Ser (0.72)                                                Asn (0.73)          His (0.71)                                                Tyr (0.61) bα Asn (0.65)                                                Pro (0.59)          Pro (0.62) bβ                                        Gly (0.53) Bα Glu (0.26) Bβ                                        ______________________________________                                    

(The letter symbols are Hα, strong α former; hα, α former; Iα; weak αformer; iα, α indifferent; bα, α breaker; and Bα strong α breaker. The βsymbols are analogous. Trp is bβ if near the C-terminal of a β-sheetregion.)

The alpha helix of a polypeptide chain comprises an average of 3.6residues per turn. In globular proteins, the average length is about 17Å, corresponding to 11 residues or 3 helix turns. In alpha and betaglobin, the helices range in length from 7 to 21 amino acids (A.A.). Thebeta pleated sheet comprises 2.3 residues per turn; the average lengthis about 20 Å or 6 residues.

Chou and Fasman define an alpha helix nucleus as a hexapeptidecontaining four helix forming residues and not more than one helixbreaker, and a beta sheet nucleus as a pentapeptide containing threebeta sheet forming residues and not more than one sheet breaker.

The amino acid sequence in the vicinity of the di-alpha linker is asfollows:

    __________________________________________________________________________    residue #                                                                            138 139                                                                              140 141     1  2   3  4                                         AA     Ser Lys                                                                              Tyr Arg                                                         (XXX).sub.n -                                                                        Val Leu                                                                              Ser Pro                                                         (SEQ ID NO:4)             (SEQ ID NO:5)                                       Helix Not                                                                            H21 HC1                                                                              HC2 HC3     NA1                                                                              NA2 A1 A2                                        Helix Pot                                                                            079 107                                                                              061 079     114                                                                              134 079                                                                              059                                       Sheet Pot                                                                            072 074                                                                              129 090     165                                                                              122 072                                                                              062                                       __________________________________________________________________________     (Note: Helix and sheet forming potentials have been multiplied by 100 for     typographical reasons.)                                                  

The di-alpha linker is preferably only 1-3 amino acids. Thus, it canform an alpha helix only in conjunction with the linker "termini". A oneor two residue linker, even if composed of amino acids with strongsecondary structure propensities, would be unlikely to assume an alphahelix or beta sheet configuration in view of the disruptive effect of,e.g., Arg 141 or Ser 3. If the linker is 3 residues long, it would bepreferable that no more than one residue be a strong alpha helix former,unless the linker also included a strong alpha helix breaker.

The amino acid sequence in the vicinity of the di-beta linker may imposemore stringent constraints.

    ______________________________________                                        143  144     145    146         1     2    3    4                             His  Lys     Tyr    His                                                       (XXX).sub.n -                                                                      Val     His    Leu  Thr                                                  (SEQ ID NO:6)       (SEQ ID NO:7)                                             H21  HC1     HC2    HC3       NA1   NA2  NA3  A1                              124  107     061    124       114   124  134  082                             071  074     129    071       165   071  122  120                             ______________________________________                                    

The di-beta linker is likely to be longer (preferably 1-9 A.A.) andtherefore more susceptible to secondary structure formation. Ifsecondary structure formation is not desired, it is desirable that theamino acid adjacent to Val-1 be an alpha helix breaker (e.g., Glycine)in view of alpha- helix propensities of Val-His-Leu. More generally, itis desirable that the linker not contain (or cooperate with theproximately linked amino acids to form) an alpha helix nucleus or betasheet nucleus.

When secondary structure is not desired, amino acids with a highpropensity toward alpha helix formation may be used in the linker ifaccompanied by "helix breaking" amino acids. Similarly, Beta sheetformation may be prevented by "sheet disrupting" amino acids.

Of course, prediction of secondary structure using Chou and Fasman'sapproach has its limitations and the ultimate test of the acceptabilityof a linker is whether or not the di- alpha or di-beta hemoglobin hasthe desired affinity for oxygen. In particular, a poly-alanine linker,despite its supposed propensity to alpha-helix formation, may well be ofvalue since the alanine group is compact and therefore the linker shouldbe quite flexible if secondary structure does not form.

In an especially preferred embodiment, di-alpha and beta globin genesare combined into a single polycistronic operon. The use of apolycistronic operon is not, however, necessary to practice the presentinvention, and the alpha (or di-alpha) and beta (or di-beta) globingenes may be expressed from separate promoters which may be the same ordifferent.

While the preferred "genetically fused hemoglobin" of the presentinvention is one comprising a di-alpha and/or di- beta globin, otherglobin chains may be genetically fused and used in the production ofmultimers of hemoglobins of species other than Hgb A1 (α₂ β₂).

Pseudo-Octameric (Ditetrameric) Hemoglobin-like Proteins With DisulfideBridges

The ability to produce pseudotetrameric recombinant hemoglobinsconsisting of a single dialpha polypeptide and two beta chains (or adibeta polypeptide and two alpha chains) provides a unique Opportunityto create an asymmetric pseudotetramer from the normally symmetricpseudotetramer. Because the two alpha globin domains are expressed as asingle polypeptide, it is possible to alter one of the alpha globindomains without altering the other. The result is a protein that, in itsfinal folded state, contains two different alpha globin domains in astrict 1:1 ratio. This type of asymmetric hemoglobin molecule, with itsunique chemical properties, cannot be easily constructed by any othermethod. A preferred embodiment of this invention would involve use ofsite-directed mutagenesis to substitute a cysteine residue in one of thetwo alpha globin domains of a di-alpha hemoglobin such as SGE1.1 (adi-alpha hemoglobin with a beta chain Presbyterian mutation also denotedrHb1.1) such that the cysteine would be on the surface of the foldedrecombinant hemoglobin molecule. A homogeneous preparation ofpseudo-octameric hemoglobin could then be formed through interhemoglobinlinkage of two pseudotetramers either directly by simple oxidation ofpurified pseudotetramers or by reaction with a bridging molecule (FIG.3).

Although direct formation of a disulfide bond between two "mono cys"tetramers is desirable in order to avoid the need for chemicalcrosslinking, naturally occurring reducing agents may reduce thedisulfide bond in vivo at a significant rate. Preliminary experimentssuggest that the rate of reduction of the bond may be influenced by thelocation of the cysteine mutation on the surface of the hemoglobin.

The surface cysteine mutants (MW=64 kDa) can be oxidized to thedisulfide-linked dimer under oxidative conditions. This can beaccomplished by stirring a concentrated solution of the expressedprotein at pH 8 under pure oxygen at 4° C. or room temperature in thedark. Trace levels of transition metal ions such as Cu⁺² may be added tolevel below 1 uM to catalyze the oxidation (1). Formation of the 128 kDaoctamer can be monitored by gel filtration. Saturation of the solutionwith oxygen at elevated pH should minimize autooxidation of recombinanthemoglobin.

An alternative procedure, which may be the preferred method ofcatalyzing this reaction, involves the use of redox buffers such asreduced and oxidized glutathione, or reduced and oxidized dithiothreitol(2). This catalysis of the reaction through disulfide interchange may benecessary to control trace transition metal catalysis (3). A second,similar approach involves conversion of the surface cysteines in the 65kDa species to sulfonates before purification (to avoid 128 kDa speciesformation during purification), followed by conversion to thedisulfide-linked 128 kDa species with reduced glutathione (2).

(1) Freedman, R. B. and Hillson, D. A. (1980) "Formation of DisulfideBonds" IN: The Enzymology of Post Translational Modification ofProteins, Vol. 1, p. 157 pp. (Academic Press).

(2) DiMarchi, R., et al. (1988) Chemical synthesis of human epidermalgrowth factor (EGF) and human type a transforming growth factor (TGFa)IN: Peptides: Chemistry and Biology (G. R. Marshall, ed.) pp. 202-203(Leiden:ESCOM).

(3) Creighton, T. E. (1978) Experimental studies of protein folding andunfolding. Prog. Biophys. Molec. Biol. 33:231-297

Multimeric Hemoglobin-Like Proteins With Other Intercysteine Linkages

It is also possible, of course, to couple two mono cys molecules with ahomobifunctional crosslinking reagent resulting in linkage vianonreducible bonds. The degree of polymerization is still controlled bythe use of the mono cys di-alpha or di-beta Hgb starting material.

By using bi-, tri-, tetra-, hexa-, or octa-functional crosslinkersseveral properties of multimeric hemoglobin which may contribute tolonger serum half life can be controlled. The crosslinkers can bedesigned to give a nonreducible bond between two tetramers, to yieldhigh molecular weight multimers of n>2 psuedo-tetramers (e.g.dodecamers, etc.) and/or to drop the overall isoelectric point of ahemoglobin octamer to further increase its half life.

Correlations of molecular weight with serum half life for proteins suchas IL-2, demonstrate that a significantly longer half life may beexpected as the molecular weight of a protein increases, particularlyabove the renal filtration limit of 50-70 kDa. However, a factorpotentially limiting the half life of multimeric hemoglobin formed by adisulfide link between tetramers is reduction of the cys-cys disulfidebond by endogenous thiol-reducing agents found in the serum. Estimatesof small molecule thiol levels in plasma vary from 17 μM to 5 μM. Themajor species is reduced glutathione. Other thiol compounds in plasmainclude cysteine, homocysteine, and gamma-glutamyl cysteine. Thus, smallmolecule plasma thiols are available for reduction of disulfide bonds.This may be reflected in the diminished half life seen withantibody-ricin A chains conjugates linked by regular disulfides (6.7hrs) relative to conjugates linked with sterically hindered, and thusless reducible, alpha-methyl disulfides (42.5 hours).

Thus, in one embodiment, the octameric hemoglobin features anonreducible sulfur-containing crosslink such as a thioether bond orthiol-maleiimide adduct. These may substantially extend the multimerhalf life. Simple homobifunctional crosslinkers or polyethylene glycol(peg) derivatives would likely be useful for this purpose (see below).The reaction of a bifunctional cysteine-specific crosslinker with amono-cys di-alpha or di-beta Hgb should limit the products of thereaction to a dumbbell-like octameric hemoglobin and unreactedhemoglobin. The reaction should be stoichiometric when the Hgb andcrosslinker are present at high concentrations and the Hgb is present ina slight excess over the crosslinker maleiimides at pH 6.5-7.0. Further,there should not be substantial interference by reaction with globinlysines. The preferential reactivity of the thiols to lysines can beroughly calculated as the product of their molar ratios and the ratio ofthe intrinsic reactivity of a maleiimide to thiols versus amines. Thisproduct is ca. 1 cys/40 lys!× 1000!=25 at pH 7. The side products wouldstill be octamers, with one attachment site being a secondary amine andthus might well be functionally equivalent to the S-crosslinkedoctamers. Hydrolysis of the maleiimide adduct at pH 7 would be slow, andthe ring opening would leave the crosslink intact.

The reaction of the thioether RC(═O)CH₂ I with the sulfhydryl-bearingprotein (R'SH) results in the crosslink RC(═O)CH₂ --S--R'. The reactionof the maleiimide with the protein results in the addition of the R'SHacross the double bond of the five-membered maleiimide ring, yielding athiomaleiimide adduct.

The following are examples of homobifunctional crosslinkers that mayform metabolically stable crosslinks between monocysteine pseudotetramers:

1) 1,2-bis-(2-iodoethoxy)ethane

2) 4,4'-dimaleiimidylbenzene or N,N'-p-phenylenedimaleiimide

3) N,N'-bis-(3-maleiimido-propionyl)-2-hydroxy-1,3-propane diamine.

Longer half lives may also be obtained by increasing the apparentsolution molecular weight by simply lengthening the distance between thetwo linked tetramers using a long crosslinking agent. The use of somepotentially novel polyethylene glycol derivatives as homobifunctionalcrosslinkers, reacting with SGE1.1 mono-cys, may provide one mechanismfor significantly increasing the molecular weight of octamerichemoglobin by virtue of the length of the crosslinker alone.

A suitable crosslinker for this purpose is

    maleiimido-CH.sub.2 CH.sub.2 C(═O)(OCH.sub.2 CH.sub.2).sub.n OC(═O)CH.sub.2 CH.sub.2 -maleiimido.

The length may be adjusted by variation of n. A few examples are givenbelow.

    ______________________________________                                        Structure     Max Length                                                                              Source                                                ______________________________________                                        n = 22         .sup.˜ 49Å                                                                   peg -1000                                             n = 76        .sup.˜ 166Å                                                                   peg -3350                                              n = 227      .sup.˜ 499Å                                                                    peg -10000                                           ______________________________________                                    

Homobifunctional N-hydroxysuccinimide-activated peg has been usedpreviously to derivatize hemoglobin. Yabuki, et al., Transfusion, 30:516(1990). This reaction resulted in a polydisperse mixture of monomeric,dimeric, and trimeric species with an average stoichiometry ofpeg/hemoglobin of 6.2. However, 83% of the hemoglobin derivatized by pegwas not crosslinked to another hemoglobin molecule. Control of thepeg-derivatization of wild-type hemoglobin was not possible becausethere is no site-directed labeling of the hemoglobin starting material.

In contrast, the combination of SGE 1.1 mono-cys starting material and apeg crosslinker should yield a substantially monodisperse dumbbell(pseudo-octameric) product. The site-direction of the crosslinkerattachment site should result in precise control of the apparentmolecular weight, which will depend on the size of the crosslinker.Moreover, careful control of the site of the cys mutation on the surfaceof the recombinant hemoglobin should ensure that the functionality ofthe derivatized hemoglobin is maintained.

Higher Multimeric Hemoglobins

The above crosslinkers all involve the attachment of one tetramer ateach end of a crosslinker. It may be advantageous to attach more thantwo tetramers to a single crosslinker to yield more oxygen-carryingcapacity and to further increase the molecular weight.

A multimeric hemoglobin may be assembled with the aid of one or morelinker peptides, each having a controlled number of reactive sites towhich a cysteine residue of a hemoglobin tetramer or pseudotetramer maybe attached, directly or indirectly.

With a peptide linker of considerable length, there is the concern thatit will be degraded by serum proteases, thus degrading the multimerichemoglobin into its component tetramers. This problem, if significant,may be remedied by use of a peptide linker which is less susceptible toproteolysis. A non-exhaustive list of such linkers would includepeptides composed of D-amino acids, peptides with stable, extended,secondary structures, and branched peptides.

In the case of peptides composed of D-amino acids, use of D-Glu or D-Aspis particularly preferred.

A number of stable, extended, secondary structures are known. Thesimplest is possibly polyproline. Another example is the 2-strandedcoiled coil, in which two peptide chains intertwine. A 4-helical or a4-stranded coiled coil are also possibilities.

Branched structures, such as those obtained by derivation of thesecondary amino group of lysine, are typically resistant to protease.

If desired, several of these approaches may be combined. For example,several coiled coils may lead off a branched structure, or D-amino acidsmay be incorporated into a coiled coil.

A hypothetical 4-tetramer coiled-coil linker complex is shown in FIG.4a. Design and synthesis of these coiled coil peptides has already beenexplored (for an example see Cohen and Parry, Proteins, 7:1-15 (1990)).The rationale for a coiled coil is that two intertwined alpha heliceswill be less sensitive to proteolytic cleavage than a single nakedsecondary structure like an extended peptide (rapidly cleaved byproteases), an alpha helix or a beta sheet.

Using molecular modeling, an internal disulfide may be designed in thecenter of a bi-functional coiled coil linker such that the strands arecovalently attached. This should stabilize formation of the correctcoiled coil crosslinker before mono-cys di-alpha or di-beta Hgb (e.g.,sge1.1 cys) is attached. Additionally, a tri-functional crosslinker canbe stabilized by use of a orthogonally-protected lysine (lys-FMOC)rather than a disulfide in the center of a proteolytically inertsecondary structure. A polyproline helix can be used as the linker, andcan be stabilized by branching the synthesis at the lys-FMOC afterremoval of the side chain. The three remaining lysines in the branchedpeptide would then be iodoacetylated to site-specifically attach athiol-reactive group using either iodoacetic anhydride orN-succinimidyliodo-acetate and subsequently reacted with sge1.1-cys. Ananalogous tetra-functional crosslinker could be synthesized by inserting1-2 prolines between two internal branching lysines to rotate them suchthat the two internal branching chains growing off the orthogonallyprotected lysines head in (nearly) opposite directions. Analogousstructures could be made using D-glutamate(E) or D-aspartate(D) toprovide protease resistance, and these would form an extendedpolyanionic chain at pH 7.

The sequence of a hypothetical alpha-helical coiled coil is modifiedfrom that given in Semchuck, et al., in Peptides: Chemistry, Structureand Biology; 566 (Rivier and Marshall, eds:1990), to leave only twolysines (K) at each end:

Ac-Lys-Cys-Ala-Glu-Leu-Glu-Gly-Arg-Leu-Glu-Ala-Leu-Glu-Gly-Arg-Leu-Glu-Ala-Leu-Glu-Gly-Arg-Leu-Glu-Ala-Leu-Glu-Gly-Arg-Leu-Glu-Ala-Leu-Glu-Gly-Lys-Leu-amide(SEQ ID NO:8)

This coiled coil should have about 10 turns of a helix and thus will beca. 54 Å long, allowing two tetramers to attach on each side withoutsteric interference. The exact sequence and length to allow appropriateplacement of 4 tetramers would depend on the results of molecularmodeling.

Suggested trifunctional and tetrafunctional crosslinkers are diagrammedbelow. ##STR1## See also FIG. 4(a).

Another possibility is an 8-hemoglobin complex (FIG. 4b). The rationalefor considering this sort of complex is that it may be the way to obtaina very long half-life, due to the extreme stability of the "crosslinker"and the substantially higher molecular weight of the complex. Thecrosslinker might take the form of a doubly branched coiled coil, with aLys(FMOC) replacing an Arg in the middle of the chain to allow thebranching, and with a polyproline helix or other protease resistantsecondary structure comprising the branching moiety. This structurecould allow attachment of 6 SGE1.1's per crosslinker. Alternatively, a4-helical bundle protein (See FIG. 4(c)) or 4-stranded coiled-coil suchas those synthesized by DeGrado, Science, 243:622 (1989), with eachhelix in the 4-helical bundle containing the consensus sequenceGly-Glu-Leu-Glu-Glu-Leu-Lys-Leu-Lys-Lys-Leu-Lys-Glu-Leu-Leu-Lye Gly,(SEQ ID NO:9) the helices being linked by three PRR or RPR loops, couldbe utilized as a suitable core for the linker. This is one of the moststable proteins known, with a G= -22 kcal/mole separating the foldedfrom the unfolded state. Each helix would be 4+ turns or ca. 22 Å long.Since this may not be enough room to fit two hemoglobins with oneanchored at each end of the helix, they might have to be attached todifferent faces of the same helix, to lysines placed at each end of thepolar face of each helix. Each helix is amphipathic; this should allowrelative freedom to have a total of 8 lysines (and no more) and tochange the remaining lysines to arginines. At least two of the i, i+4salt bridges per helix would be retained for stability of the protein.Attachment of an externally crosslinkable cysteine-bearing tetramercould be via iodoacetylation of the lysine epsilon amino groups and thenreaction with the thiol group of the cysteine.

An example of a modification that might allow more room betweentetramers would be addition of one turn of the helix to the N-terminusof the A and C helices and the C- terminus of the B and D helices. Thisand similar modifications would be subject to modeling and experimentalconstraints.

Analogous core proteins could be made as mutants of known 4-helicalbundle proteins such as myohemerythrin or apoferritin, with the surfaceresidues changed so that 8 (or more if topologically possible) lysines(2 per helix) exist on the surface for subsequent modification andattachment of the tetramer. Poly(tetrameric) Hemoglobins with ReducedIsoelectric Points

If the isoelectric point of the whole crosslinked conjugate also affectsthe serum half life, via electrostatic exclusion from the renal filter's"pore", additional negative charges could be included in the crosslinkitself (rather than in the hemoglobin, which could change the functionof the molecule) to drop the isoelectric point of the overallcrosslinked particle. An additional benefit of this might be reduceduptake by the reticuloendothelial system, this uptake being a functionof pI for cationized albumin.

We have preliminary evidence from succinylation of SGE1.1 whichcorrelates the number of modified lysines with isoelectric point. Thisgives a rough estimate of the number of lys to glu and/or lys to aspmutations which may be necessary to reach a pI of 5 or less, the pIrange which we expect we need to significantly extend half life. Webelieve that as many as 8 lysines may have to be modified (a total shiftin charge of 16 units) to drop the pI roughly 2 units. It should be lessdisruptive of the functional properties of hemoglobin to do this via apeptide crosslinker rather than by mutation of the alpha and beta globinsubunits proper. However, some mutations could be made in thecrosslinker and the remainder in the subunits. As before, the SGE1.1-cyswould be attached to iodoacetylated lysine epsilon amino groups byreaction at pH6.5-7.0.

For human serum albumin in the rat, the half life varied roughlylinearly with the pI of the protein, from ca. 4.6 hours for nativealbumin (pI=4) to 0.8 hrs at a pI above 9.5. Clearance was probably bymultiple mechanisms, including potentially increased uptake into thereticuloendothelial system with increased pI. For rat trypsinogens, thedifference in serum half life between versions with a pI of 5.-0(t_(1/2) of 4 min) was even larger. Thus a lower pI clearly appears tobe an important variable in the serum half life of these proteins.

The following table gives examples of crosslinkers between mono-cystetramers which should diminish the isoelectric point of the overallcomplex.

    ______________________________________                                        Source  Sequence                                                              ______________________________________                                        polyasp or polyglu                                                                     ##STR2##          n probably ≧ 10-12, X = D or E.sup.-.       polyasp or polyglu                                                                     ##STR3##          n > 2 to provide flexibility at each terminus,                                m ≧ 10-12, X = D.sup.-  or E.sup.-          polyasp or polyglu                                                                     ##STR4##          n ≧ 5-6, m ≧ 10-12, X = D.sup.-                                 or E.sup.-                                         ______________________________________                                    

A number of the proposed crosslinkers could combine at least two, orpossibly three of these attributes for potential additive effects.

It is possible that the unique amine groups in the peptide crosslinkerscould be directly iodoacetylated during the peptide synthesis bytreating iodoacetic acid as the last amino acid to be added, afterdeprotecting the lysine amine groups on the resin. In this case, thelysines would be orthogonally protected with N-FMOC orN-nitropyridinesulfenyl groups, or with BNPEOC. This could greatlysimplify their synthesis.

Alternate methodologies to iodoacetylation as part of the synthesiscould include the reaction of either sge1.1 -SH or the peptidecrosslinker with a heterobifunctional crosslinker specific forsulfhydryls and amines, such as sulfo-SMCC or similar reagents availablefrom Pierce Chemical Co. (Rockford, Ill.).

Genetically Fused Poly(tetrameric) Hemoglobins

Another approach to the preparation of multimeric (e.g., polytetrameric)hemoglobin involves the genetic fusing of individual tetramers utilizingother linkers. Two or more tetramers may be linked, depending on thedesired molecular weight and the efficiency of folding of the finalmolecule. The dialpha (or dibeta)subunits from different tetramers of adi- alpha or di-beta Hgb might be genetically fused together into anextended, polypeptide which would link the individual pseudotetramericdomains.

Proteolytically stable extended polypeptide linkages can be envisioned.Desirable linker features might include 1) a number of glycines at eachend to allow flexibility in entering the dialpha (or beta) terminaldomains, and to decouple the linker secondary structure from that of thedialpha (or beta) terminal domains; 2) stiffness to separate tetramers,obtainable by an extended structure such as a polyproline helix or bypolyglutamate or polyaspartate; and 3) inertness to proteases (videsupra or as in a collagen sequence). Several examples of such sequencesare listed below. Obviously any other of the peptide linkers mentionedin this specification could be tried after first sterically modeling thefused-dialpha (or dibeta) termini environment. The links would go fromthe C-terminus of one dialpha to the N- terminus of the next and wouldbe synthesized as a single gene. Besides modeling segments ofprotease-resistant or negatively charged secondary structure, one ormore of the Artemia linkers should be modeled between tetramers. Thebeta chains could also be joined in this fashion, although the resultsof this on protein function would be unknown. It might be feasible tomake an intermolecular di-beta (sge1.1) with or without additionalintrachain crosslinkages.

    ______________________________________                                        Source   Sequence                                                             ______________________________________                                        polyproline                                                                            di α or β C term-(G)n--(P)n--(G)n-di                      helix    α or β N terminus n probably ≧ 3,                           m probably ≧ 10-12                                            polyaspartate                                                                          --(G)n--(D)n--(G)n--                                                 or glutamate                                                                           (should drop pI of complex)                                          Artemia linker                                                                         --(G)n--Leu--Arg--Arg--Gln--Ile--Asp--Leu--                          (example)                                                                              Glu--Val--Thy--Gly--Leu--(G)n--; n ≧ 0                                  SEQ ID NO:30!                                                       a helical                                                                              --(G)n--Lys--Cys--Ala--Glu--Leu--Glu--Gly--                          coiled coil                                                                            Lsy--Leu--Gly--Ala--Leu--Glu--Gly--Lys--Leu--                                 Glu--Ala--Leu--Glu--Gly--Lys--Leu--Glu--Ala--                                 Leu--Glu--Gly--Lys--Leu--Glu--Ala--Leu--Glu--                                 Gly                                                                           <-- not fused to terminus (should                                             form octamer with coiled-coil crosslink)                                       SEQ ID NO:31!                                                       ______________________________________                                    

We have determined the minimum of the intertetramer linker as follows.Two structures of human hemoglobin A_(o) (either both in the oxy form orboth in the deoxy form) taken or assembled from the Brookhaven ProteinData Bank were docked as close together as possible without van derWaals overlap between any residues, using the program Insight (Biosym.Inc., San Diego, Calif.) The distance from the alpha chain C terminalresidue arg 141 to the amino terminal nitrogen of the alpha chain Nterminal residue val 1 (in one structure) was then measured. Thisdistance was ca. 22 Å when both molecules had the oxy structure and ca.18 Å when both were in the deoxy structure. In the oxy and deoxystructures, the valine at the alpha chain N terminus is exposed at theside of a cleft in the structure, while the arg carboxylate is at thebottom of the cleft. Thus it is possible to genetically fuse these twotermini without suffering a large structural displacement of residuesaround either terminal amino acid. A suitable intertetramer linker willbe at least 18-22 angstroms long, preferably longer in order to give thestructure additional flexibility. There is no fixed upper limit on thelength of the linker, however, the longer the linker, the moresusceptible it is to protease, and, if the molecule appears largeenough, it may be phagocytosed by macrodhages of the reticuloendothelialsystem. A few examples of suitable linkers are listed below.

An alternative fusion may be envisioned between a truncated alpha chainin one hemoglobin and the N terminal alpha val 1 in the secondhemoglobin. The first molecule could be truncated at ser 138, whichintermolecular N terminal to C terminal distance is about 17 Å (deoxy)and 22 Å (oxy), and examples of genetically inserted linkers spanningthis distance are listed below.

Thus two hemoglobin molecules could be linked (by fusing twointermolecular alpha domains) to generate a fusion protein approximatelytwice the size of normal human hemoglobin. An additional intramolecularcrosslink, as introduced into rHb1.1 to prevent dissociation ofhemoglobin into dimers, could be included as well, giving a fusion offour alpha domains.

We expect that the genetically inserted links will be stable in thepresence of proteases, due to the steric occlusion by the twohemoglobins surrounding the linkage. This resistance may be furtherenhanced by the use of glycines, bonds between which may be lesssusceptible to proteases, since most proteases have side chainspecificity for residues other than glycine (which has only a hydrogenas a sidechain, and thus; may result in a poor Km of this substrate fora protease). A polyproline helix may also be used as a linker to enhancestability to proteases. Fusion of a polyglutamate or polyaspartate as alinker might allow a much lower isoelectric point for the complex, andthus a longer serum half life.

    ______________________________________                                        Intertetrameric Linkers for Inclusion in                                      Pseudooligomeric Polypeptides                                                             end-to-end                                                                              conforma-                                               Linker      Distance  tion      Comments                                      ______________________________________                                        (gly).sub.7 -                                                                             25Å   extended  minimal length                                (SEQ ID NO:32)                  for gly linker to                                                             span termini in                                                               both oxy and                                                                  deoxy structures.                                                             Longer linkers                                                                (up to 20-50                                                                  residues) may                                                                 also work                                                                     favorably.                                    (gly).sub.1-3 (ala).sub.12 -                                                              20Å-  Ala in    the Gly are added                             (gly).sub.1-3 -                                                                           40Å   right     for flexibility                               (SEQ ID NO:33)        handed    and minimal                                   helix                 alpha     disturbance of Hb                                                   helix     structure around                                                              their fusion with                                                             the N and C.                                                                  termini. Length                                                               is dependent on                                                               the number of                                                                 glycines and the                                                              degree of                                                                     extension                                     (gly).sub.1-3 (pro).sub.12-16 -                                                           21-       pro in a  12, 14, 16                                    (gly).sub.1-3 -                                                                           48Å   left      prolines. Length                              (SEQ ID NO:34)        handed    dependent on                                  proline               poly-     number of                                     helix                 proline   prolines and                                                        helix     glycines                                      (gly).sub.1-3 -                                                                           26-                 Asp residues add                              (asp).sub.1-30 -                                                                          49Å             negative charges                              (gly.sub.1-3)-                                                                (SEQ ID NO:35)                                                                ______________________________________                                    

Other residues could be substituted into these linkers while leavingtheir length essentially the same, including complete linkers taken fromthe sequence of other known human proteins such as hemoglobin, toprevent any recognition of the multimer as a foreign protein.

Use of linkers with a maximal length more than 18 Å and less than 22 Åmay differentially stabilize the deoxy structure, and may result in alowered oxygen affinity for the multimer.

Octameric Hemoglobins Formed Without Use of an Pseudooligomeric Globin

It is possible to produce an octameric hemoglobin, without substantialproduction of higher multimers, by suitable cysteine mutation of eitherthe alpha or beta chain (see FIGS. 5a-5c).

Hemoglobin mutants containing one X to cys mutation in the beta chaingene (giving two per tetramer) or in the alpha chain gene (also givingtwo per tetramer), in which the residues mutated to cysteine are both onor very close to the surface of the subunit and are as close to the dyadaxis separating the subunits, may form octamers (two hemoglobins) linkedby two disulfides. Polymerization of such mutants should be retarded bythe proximity of the two disulfides to each other, such that after onedisulfide is formed, a third incoming hemoglobin will be stericallyhindered from reacting with either free cysteine on the two originalhemoglobins.

Because it is possible that this mutant may form higher order polymers(rather than simply the octamer), a diluted solution may be used invitro for formation of disulfide bonds. The kinetics of polymerizationof hemoglobin should be at least second order (or a higher order) inhemoglobin concentration, while after one disulfide is formed, theformation of the second disulfide between two tetramers should be zeroorder in hemoglobin. Thus the ratio of polymerized product to octamershould diminish as the hemoglobin concentration is decreased. Ifformation of octamers is done under oxygenated conditions, the yield ofoctamers vs. polymers may increase further, since the distance betweenthe two cys mutations is less in every case in the oxy hemoglobinstructure than in the deoxy structure.

A list of preferred mutation sites in both the beta chain and the alphachain is provided below: Beta and alpha chain mutation sites for x tocys mutations to form disulfide-bond linked octameric hemoglobin.

    ______________________________________                                        Chain/    Old       New                                                       Mutation  Distance (Å)                                                                        Distance (Å)                                                                          Comment                                       ______________________________________                                        beta Asn  22        18          no listed                                     80 to cys                       deleterious                                                                   mutations, asn 80                                                             is on surface                                 beta Asp  24        22          Hb Tampa.sup.a (asp to                        79 to cys                       tyr) has no major                                                             abnormal property                                                             listed; Hb G-His-                                                             Tsou (asp to gly)                                                             has increased O.sub.2                                                         affinity; is on                                                               surface                                       alpha Asn 24        20          on surface; no                                78 to cys                       major.sup.a abnormal                                                          properties of                                                                 known mutations                                                               of asn 78                                     alpha Asp   22A     18          on surface; no                                75 to cys                       major abnormal                                                                properties of                                                                 known mutations                                                               of asp 75                                     alpha Asp 26        20          on surface; no                                74 to cys                       major.sup.a abnormal                                                          properties of                                                                 known mutations                                                               of asp 74                                     ______________________________________                                         .sup.a R. N. Wrightstone. Policies of the International Hemoglobin            Information Center (IHIC), Comprehensive Sickle Cell Center, Medical          College of Georgia. 1988.                                                

Gene Construction and Expression

The DNA sequences encoding the individual polypeptide chains may be ofgenomic, cDNA and synthetic origin, or a combination thereof. Since thegenomic globin genes contains introns, genomic DNA must either beexpressed in a host which can properly splice the premessenger RNA ormodified by excising the introns. Use of an at least partially syntheticgene is preferable for several reasons. First, the codons encoding thedesired amino acids may be selected with a view to providing unique ornearly unique restriction sites at convenient points in the sequence,thus facilitating rapid alteration of the sequence by cassettemutagenesis. Second., the codon selection may be made to optimizeexpression in a selected host. For codon preferences in E. coli, seeKonigsberg, et al., PNAS, 80:687-91 (1983). Finally, secondarystructures formed by the messenger RNA transcript may interfere withtranscription or translation. If so, these secondary structures may beeliminated by altering the codon selections.

Of course, if a linker is used to genetically crosslink subunits, thelinker will normally be encoded by a synthetic DNA.

The present invention is not limited to the use of any particular hostcell, vector, or promoter. The host cell may be prokaryotic oreukaryotic, and, in the latter case, may be a plant, insect or mammalian(including human) cell. The cell may also be of any suitable tissuetype, including, inter alia, an erythrocyte. However, the preferred hostcells are bacterial (especially, E. coli) and yeast (especially S.cerevisiae) cells. The promoter selected must be functional in thedesired host cells. It preferably is an inducible promoter which, uponinduction, provides a high rate of transcription. A preferred bacterialpromoter is the Tac promoter, a trp/lac hybrid described fully inDeBoer, U.S. Pat. NO. 4,551,433 and commercially available fromPharmacia-LKB. Other promoters which might be used include thetemperature sensitive lambda P_(L) and P_(R) promoters, as well. as thelac, trp, trc, pIN (lipoprotein promoter and lac operator hybrid), galand heat shock promoters. The promoter used need not be identical to anynaturally-occurring promoter. Guidance for the design of promoters isprovided by studies of promoter structure such as that of Harley andReynolds, Nucleic Acids Res., 15:2343-61 (1987) and papers citedtherein. The location of the promoter relative to the first structuralgene may be optimized. See Roberts, et al., PNAS (U.S.A.), 76:760-4(1979). The use of a single promoter is favored. Suitable yeastexpression systems are described in detail elsewhere in thisspecification.

The vector used must be one having an origin of replication which isfunctional in the host cell. It desirably also has unique restrictionsites for insertion of the globin genes and the desired regulatoryelements and a conventional selectable marker. A vector may be modifiedto introduce or eliminate restriction sites to make it more suitable forfuther manipulations.

The component polypeptide chains of the multimeric hemoglobin may beexpressed either directly or as part of fusion proteins. When expressedas fusion proteins, the latter may include a site at which they may becleaved to release the globin-related moiety free of extraneouspolypeptide. If so, a site sensitive to the enzyme Factor Xa may beprovided, as taught in Nagai and Thorgenson, EP Appl 161,937,incorporated by references herein. Alternatively, the fusion proteinsmay be synthesized, folded and heme incorporated to yield a hemoglobinanalogue. The direct expression of the component polypeptides isdesirable.

In bacterial mRNA, the site at which the ribosome binds to the messengeris a polypurine stretch which lies 4-7 bases upstream of the start (AUG)codon. The consensus sequence of this stretch is 5' . . . AGGAGG . . .3', and is frequently referred to as the Shine-Dalgarno sequence. Shineand Dalgarno, Nature, 254: 34 (1975). The exact distance between the SDsequence and the translational start codon, and the base sequence ofthis "spacer" region, affect the efficiency of translation and may beoptimized empirically. Shepard, et al., DNA 1: 125 (1985); DeBoer, etal., DNA 2: 231 (1983); Hui, et al., EMBO J., 3: 623 (1984).

In addition, the SD sequence may itself be modified to alter expression.Hui and DeBoer, PNAS (U.S.A.), 84:4762-66(1987). Comparative studies ofribosomal binding sites, such as the study of Scherer, et al., NucleicAcids Res., 8:3895-3907 (1980), may provide guidance as to suitable basechanges. If the hemoglobin is to be expressed in a host other than E.coli, a ribosomal-binding site preferred by that host should beprovided. Zaghloul and Doi, J. Bacteriol., 168:1033-35 (1986).

Any host may be used which recognizes the selected promoter andribosomal binding site and which has the capability of synthesizing andincorporating heme. Bacterial and yeast hosts are preferred.

The intracellularly assembled hemoglobin may be recovered from theproducing cells and purified by any art- recognized technique.

Polycistronic Expression in Bacteria

While not required, it is desirable that the subunits, when expressed inbacteria, be co-expressed in the same cell, and it is still morepreferable that they be co-expressed polycistronically. A polycistronicoperon encodes a single messenger RNA transcript having one promotersequence, but two or more pairs of start and stop codons that definedistinctly translatable sequences. Each such sequence is known as a"cistron," and the polypeptides corresponding to the cistrons are thusco-expressed under the control of the single promoter.

The majority of bacterial operons are polycistronic, that is, severaldifferent genes are transcribed as a single message from their operons.Examples include the lactose operon with three linked genes (lac Z, lacY and lac A) and the tryptophan operon with five associated genes (trpE, trp D, trp C, trp B, and trp A). In these operons, the synthesis ofmessenger RNA is initiated at the promoter and, within the transcript,coding regions are separated by intercistronic regions of variouslengths. (An operon is a cluster of genes that is controlled as a singletranscriptional genetic unit). Translational efficiency varies fromcistron to cistron. Kastelein, at al., Gene, 23: 245-54 (1983).

When intercistronic regions are longer than the span of the ribosome(about 35 bases), dissociation at the stop codon of one cistron isfollowed by independent initiation at the next cistron. With shorterintercistronic regions, or with overlapping cistrons, the 30S subunit ofa terminating ribosome may fail to dissociate from the polycistronicmRNA, being instantly attracted to the next translational initiationsite. Lewin, Genes, 143-162 (John Wiley & Sons: 1977).

Unlike bacterial mRNAs, eukaryotic mRNAs are generally monocistronic innature. Lewin, Gene Expression, 157.

In one embodiment, expression of the genes encoding two or morecomponent polypeptides are driven by a single promoter, and the genesare arranged so that a polycistronic messenger RNA transcript istranscribed, from which the separate globin-like polypeptides aresubsequently translated. However, the present invention includes theco-expression of the different polypeptides from separate promoters,i.e., the host transcribes separate alpha and beta globin mRNAs.

Ideally, alpha and beta globin-like domains are expressed instoichiometrically equal amounts. While use of a single promoter doesnot guarantee equality, it eliminates one unbalancing influence--differences in transcription owing to differences in promoter strengthand accessibility. If differences in promoter strength were minimized byuse of two identical promoters on the same plasmid, plasmid stabilitywould be reduced as there would be a propensity toward recombination ofthe homologous regions.

Preferably, the alpha and beta globin-like domain-encoding genes arearranged so that the ribosome will translate the alpha globin cistronsfirst. The rationale is that there is some basis for believing thatalpha globin affects the folding of beta globin. Nonetheless, theposition of the genes may be switched so that a beta globin-like domainis synthesized first.

The stability of the polycistronic mRNA transcript, the efficacy of itstranslation into alpha and beta globin-like polypeptides, and thefolding of the globin chains into tetrameric hemoglobin may be modifiedby varying the length and base sequence of the intercistronic regions(the region lying between the stop codon of one cistron and the startcodon of the next cistron), the phasing of a second cistron relative toa first cistron, and the position and sequence of the ribosomal bindingsite for the one cistron relative to the preceding cistron.

In a preferred embodiment, the alpha and beta globin-like polypeptidesgenes are each preceded by a short "introductory" cistron or "ribosomalloader" which facilities the subsequent translation of the globincistron. In FIG. 2, region A (FIGS. 2athrough 2c) contains two cistronsand a Shine-Dalgarno sequence preceding each cistron. The firstShine-Dalgarno sequence (SD#1) is bound by the ribosome, which thentranslates the first cistron, a short. cistron encoding an octapeptide.(This cistron is referred to as an "introductory cistron or ribosomalloader.) The second cistron is a globin gene, in this case, an FXalpha-globin gene. The Shine-Dalgarno sequence (SD#2) for facilitatingtranslation of the second cistron actually lies within the firstcistron. For this reason, the two are said to be "translationallycoupled". Region B (FIGS. 2c through 2e) is identical in structure,except that the second cistron encodes FX-beta globin. Between regions Aand B is a 43-base intercistronic region (FIG. 2c). The introductorycistrons of regions A and B correspond to the first cistron of thetwo-cistron expression system denoted pCZ144 in Schoner, et al., Meth.Enzymol., 153: 401-16 (1987). The present invention is not, however,limited to the particular "starter" cistron taught by Schoner, et al.;other introductory cistrons that allow for restart of high leveltranslation of a following cistron may be used.

Guidance as to the design of intercistronic sequences and as to thelocation of SD sequences may be obtained by comparing the translationalefficiency of spontaneous or controlled mutants of the samepolycistronic operon, as exemplified by Schoner, et al., PNAS, 83:8506-10 (1980). It is also possible to look for consensus features inthe intercistronic regions of different operons. McCarthy, et al., EMBOJ., 4: 519-26 (1985) have identified a translation- enhancingintercistronic sequence in the E. coli atp operon.

The present invention is intended to reduce or avoid the localization ofthe hemoglobin or its component polypeptides into inclusion bodies.Consequently, a further feature of the invention is that the functionalhemoglobin is substantially found (preferably over 80%) in the solublefraction of the cell. It appears that with this invention, over 90% ofthe functional hemoglobin can be so directed when alpha₂ beta₂hemoglobin is assembled from alpha- and beta- globin chains co-expressedfrom a tetracistronic operon as described herein. With di-alpha, beta₂hemoglobin, nearly 100% is soluble when expression is induced at 25° C.and less at higher induction temperatures. These percentages reflect thepercent of all di-alpha and beta chains found in the soluble fraction ofthe cell and not actual recovery of protein from the cell.

Expression in Yeast

In another embodiment the present invention relates to the production ofhemoglobin-like molecules in yeast. Our preferred host for expression inyeast is Saccharomyces cerevisiae. However, other fungi or yeast may beused for the purpose, such as strains of Aspergillus or Pichia. Foryeast to be a suitable host it must be capable of being transformed withrecombinant vectors, either replicating or integrating types. Thisallows the insertion of the desired DNA sequence for the gene ofinterest. It must also be capable of high density cell growth, inappropriate volume to provide sufficient cell mass to isolate thedesired gene product from the desired reaction vessels, where ideallythe growth would be easily controlled by several parameters includingnutrient formulation, agitation and oxygen transfer and temperature. Itis also desirable to be able to induce the expression of proteinsynthesis with the manipulation of the media, temperature, or by theaddition or consumption of certain chemicals. Finally, to be a suitablehost, the yeast must be capable of producing recombinant proteins,preferably in excess of 1% of the total cell protein. This allows morefacile isolation of the desired recombinant protein.

Either a haploid or a diploid strain of S. cerevisiae may be used. Forexample, the following diploid strains are preferred:

BJY3505×RSY330

BJY3505×BJY 1991

Other matings may likewise be used in practicing the present invention.

The use of protease-deficient strains may also be advantageous.

Yeast expression systems can be divided into two main categories: (1)Systems designed to secrete protein and (2) system designed for thecytoplasmic expression of proteins. At present, cytoplasmic expressionis preferred since the yeast cells fold together the globin chains andincorporate heme to produce hemoglobin in vivo. However, it is possibleto separately express and secrete the alpha and beta globin chains andassemble hemoglobin in vitro.

The globin genes must be placed under the control of a suitablepromoter. The commonly used yeast promoters generally fall into twobroad categories: regulated and constitutive. Constitutive promotersthat are in wide use include GAP, PGK (phosphoglycerate kinase) and theα-factor promoter. Regulated promoters have also been used and theseinclude the yeast metallothionein promoter (regulated by copper), theGall-10 promoter, GAL7 promoter (regulated by galactose and glucose) theADHII promoter (regulated by ethanol and glucose) the PH05 promoter(phosphate regulation) and several hybrid promoters such as PH05-GAP,GAL-PGK, ADHII-GAP, and GAL-CYC1.

The use of a GAL-GAP hybrid promoter is preferred. Both elements (theGA_(USA) and the GAP transcriptional initiation site) are wellunderstood. Studies on the mechanisms of transcriptional regulation ofthe GAL regulon have been fairly extensive. The galactose regulonincludes five genes that encode enzymes required for the utilization ofgalactose. Four of these genes (GAL1, GAL7, GAL10, and GAL2) areexpressed only in the presence of galactose. Galactose induction doesnot occur in the presence of glucose unless the yeast strain bears amutation in the REG1 gene. The GAL1, 7, 10 and 2 genes are regulated byat least two other genes, GAL80 and GAL4. The GAL4 gene is atranscriptional activator protein that activates mRNA synthesis from theGAL1, 7, 10 and 2 upstream activator sequences (UAS_(GAL)). AlthoughGAL4 is constitutively expressed, it is functionally silent in theabsence of galactose. Repression of GAL4 activity, in the absence ofgalactose is maintained by the product of the GAL80 gene. The GAL80protein apparently interacts physically with GAL4 to preventtranscriptional activation. Presumably galactose or a galactosederivative prevents this interaction to allow GAL4 mediated induction.

Haploid strains of S. cerevisiae have three different genes encoding theenzyme glyceraldehyde-3-phosphate dehydrogenase (GAP). These genes havebeen designated TDH1, TDH2 and TDH3 and each is present as a single copyper haploid genome. The TDH3 gene produces approximately 60% of thecell's GAP enzyme and TDH1 and 2 produce about 12% and 28%, respectively(McAllister, L and M. J. Holland, 1985. J. Biol Chem, 260: 15019-15027).Holland's group (Holland et al. 1981. J. Biol Chem, 256:1385-1395; andHolland et al. 1983. J Biol Chem 258:5291-5299) has cloned andcharacterized the three GAP genes of S. cerevisiae. The clones have beendesignated pGAP11, pGAP63, and pGAP491. pGAP491 corresponds to the TDH3gene and is therefore, the most highly expressed.

This promoter is commonly used as a 600-850 bp fragment and isessentially un-regulated. In its long form this is a very powerfulpromoter. The form we are using consists of only ˜200 bp 5' of thetranslational initiation site. This form, with no added enhancersequences is substantially less active than the longer form of thepromoter (Edens, L. et al. Cell, 37:629 (1984)). Our addition of the GALenhancer region confers both regulation and high levels of expression.With only the GAP491 promoter, alpha and beta globin were produced at alevel of less than 0.2% total cell protein; with the GAL-GAP491 hybridpromoter, expression jumped to 7-10% total cell protein.

Several other hybrid promoters are of particular interest: GAL-SIGMA;SIGMA-GAP; GAL-EF III; SIGMA-EF III.

One could easily conceive of other promoter systems that would alsowork. This would include, but not be limited to, a variety ofconstitutive promoters. For example, the yeast mating factorα (MFα)promoter or the mating factor a promoter MF(a), the phosphoglyceratekinase promoter (PGK), hexokinase1, hexokinase2, glucokinase, pyruvatekinase, triose phosphate isomerase, phosphoglycerate isomerase,phosphoglycerate mutase, phosphofructose kinase or aldolase promotersmay all be used. In short, any well expressed yeast promoter may workfor expression of hemoglobin in yeast. A wide variety of naturallyoccurring, regulated promoters could also be used, for example: GAL1-10,GAL7, PH05, ADHII have all been used to produce heterologous proteins inyeast. A variety of synthetic or semi-synthetic yeast promoters couldalso be employed such as GAL-PGK, GAL-MFα-1, GAL-MFa1, GAL-SIGMA. ADHIIregulatory sequences could also be coupled to strong transcriptionalinitiation sites derived from a variety of promoters. The PHO5regulatory sequence or the sigma element regulatory sequences could alsobe used to construct powerful hybrid promoters. In addition to yeastpromoters, it is conceivable that one could use a powerful prokaryoticpromoter like the T7 promoter. In this case, one could place the T7polymerase under the control of a tightly regulated yeast promoter.Induction of the phage polymerase in yeast cells bearing hemoglobingenes under T7 promoter regulation would allow transcription of thegenes by this very efficient phage polymerase.

Because most of the yeast regulatory sequences described above serve astargets for proteins that are positive regulators of transcription, itis conceivable that these proteins may limit transcription in situationswhere the target sequence is present in many copies. Such a situationmay obtain with vectors such as pC1B, pCIT, pC1U or pC1N which may bepresent in excess of 200 copies per cell. Over-expression of thepositive regulator (for example GAL4) may result in enhanced expression.It is possible to construct a strain in which the GAL4 gene is alteredto remove its promoter and the promoter replaced with the GAL7 orGAL1-10 promoters, both of which are transcribed more efficiently thanthe GAL4 promoter. In this situation, the positive transcriptionalactivator protein GAL4 would be expressed at elevated levels at the timehemoglobin expression was induced.

The consensus sequence for higher eukaryotic ribosome binding sites hasbeen defined by Kozak (Cell, 44:283-292 (1986)) to be: G^(AA) _(G)CCAUGG (SEQ ID NO:10). Deviations from this sequence, particularly atthe -3 position (A or G), have a large effect on translation of aparticular mRNA. Virtually all highly expressed mammalian genes use thissequence. Highly expressed yeast mRNAs, on the other hand, differ fromthis sequence and instead use the sequence AAAAAUGU (Cigan and Donahue,Gene, 59:1-18 (1987)). The ribosome binding site that we use forexpression of the α and β-globins corresponds to the higher eukaryoticribosome binding site. It is within the contemplation of this inventionto systematically alter this RBS to test the effects of changes thatmake it more closely resemble the RBS of yeast. It should be pointedout, however, that alterations at the -2, -1 and +3 positions, ingeneral, have been found to only slightly affect translationalefficiency in yeast and in mammals.

Intracellular expression of genes in S. cerevisiae is primarily affectedby the strength of the promoter associated with the gene, the plasmidcopy number (for plasmid-borne genes), the transcription terminator, thehost strain, and the codon preference pattern of the gene. Whensecretion of the gene product is desired, the secretion leader sequencebecomes significant. It should be noted that with multicopy plasmids,secretion efficiency may be reduced by strong promoter constructions.Ernst, DNA 5:483-491 (1986).

A variety of extrachromosomalty replicating vectors (plasmids) areavailable for transforming yeast cells. The most useful multicopyextrachromosomal yeast vectors are shuttle vectors that use a fulllength 2μ-circle combined with an E. coli plasmid. These vectors carrygenes that allows one to maintain the plasmid in appropriate yeastmutants and antibiotic resistance markers that allow selection in E.coli. Use of the full-length 2μ-circle, in contrast to vectorscontaining only a partial 2μ sequence, generally results in much higherplasmid stability, particularly in yeast strains that have been cured ofendogenous 2μ plasmid. The pC series of vectors described herein arevectors of this type.

Strains could also be constructed in such a way that the GALGAPhemoglobin expression cassettes were integrated into chromosomes byusing yeast integrating vectors. Although the copy number of thehemoglobin genes would be lower than for plasmid vectors, they would bequite stable and perhaps not require selection to be maintained in thehost cell. Yeast integrating vectors include Yip5 (Struhl, et al, PNAS,76:1035-39, 1979), Yipl (Id.), and pGT6 (Tschumper and Carbon, Gene,10:157-166, 1980). For information on these and other yeast vectors, seePouwels, et al., Clonin Vectors, A Laboration Manual, (Elsevier, 1985).

The genes encoding the desired globin-like domains may be introduced byseparate plasmids, or both upon the same plasmid.

Highly expressed yeast genes show a very high codon bias. The genesencoding glyceraldehyde-3-phosphate dehydrogenase and ADH-I, forexample, show a 90% bias for a set of 25 codons. Highly expressed yeastgenes (>1% of the total mRNA) have yeast codon bias indices of >0.90.Moderately expressed genes (0.1-0.05% of the total mRNA) have biasindices of 0.6-0.8, and genes expressed at low levels (>0.05% of thetotal cell protein) have a codon bias of 0.10-0.50 (Bennetzen and Hall,J. Biol. Chem., 257:3026-3031 (1982)). The calculated value for thecodohs of the human α-globin cDNA is 0.23. A similar value can becalculated for the β-globin cDNA. Because there is a very highcorrelation between the most commonly used codons, it is possible thathemoglobin expression from the human cDNA in yeast may be limited by theavailability of the appropriate tRNA molecules. If this is so, acomplete synthesis of the gene using the most highly favored yeastcodons could improve the expression levels. It is quite possible thatthe greatest negative effect of adverse codon use would be if there wasan abundance of codons used in the cDNA that are represented by lowabundance tRNAs. In such a case, high level expression of hemoglobincould completely drain that pool of tRNA molecules, reducing translationnot only of hemoglobin but of yeast proteins that happen to use thatcodon as well. In the case of the α-globin human cDNA, the most commonlyused leucine codon is CTG (14 of 21), this codon is never used in highlyexpressed yeast genes (Guthrie and Abelson, The Molecular Biology of theYeast Saccharomyces, Eds. Stratern, Jones and Broach, 1982. Cold SpringHarbor, N.Y.). The low codon bias index and the presence of rare yeastcodons in the globin cDNAs have been sufficient incentive for us tosynthesize a modified form of the alpha- and beta-globin genes using thepreferred yeast codons.

Miscellaneous

The appended claims are hereby incorporated by reference as a furtherenumeration of the preferred embodiments. All cited references areincorporated by reference to the extent necessary to enable the practiceof the invention as now or hereafter claimed.

Preparation of expression vectors suitable for use in production of theclaimed multimeric hemoglobins may be facilitated by the Budapest Treatydeposit of the following vectors, all made with American Type CultureCollection, Rockville, Md. U.S.A. on May 10, 1990:

ATCC 68323 pDL III-14c

This is a derivative of pKK223-3 (Pharmacia LKB, Piscataway, N.J.,U.S.A.) and pGEM1 (Promega Corp., Madison, Wis., U.S.A.) which carriessynthetic genes for des-Val alpha globin and des-Val beta globin as partof a polycistronic operon driven by a single Tac promoter.

ATCC68324 pDL IV-8a

This is a derivative of PDL III-14c which contains a fused gene encodingan alpha globin moiety, a glycine, and a second alpha globin moiety, aswell as a second gene encoding des-Val beta globin.

ATCC 20992 pGS 389

This is a yeast vector which expresses alpha and beta globin undercontrol of GALGAP promoters.

The deposit of these vectors should not be construed as a license tomake, use or sell them.

EXAMPLE 1

Construction of di-α globin mono-cysteine (A71C, D75C, or S81C) mutantexpression vector

The following plasmids, whose preparation is fully described in Hoffman,et al., WO88/09179, were manipulated in this Example.

Plasmid pDL II-83a

A gene encoding a Met initiation codon, a Factor X site(Ile-Glu-Gly-Arg)(SEQ ID NO:11), and human alpha globin, collectivelyreferred to as FX-A, was synthesized and cloned into the XmaI/PstI sitesof M13mp19. The EcoRI-PstI fragment bearing the FX-A gene was excisedand recloned into pKK 223-3, placing it under control of the Tacpromoter. This derivative was called pDLII-62m. The FX-A gene wasremoved from pDLII-62m and ligated with EcoRI/PstI linearized pGEM1,forming pGEM-FX-A. This was digested with NdeI and EagI, removing theFactor Xa coding sequence (and part of the α globin coding sequence).The excised fragment was replaced by a synthetic oligonucleotide whichrestored the missing α globin codons; the resulting plasmid was namedpDLII-83a. The protein expressed was "Met-Val-Leu- . . . "

Plasmid pDLII-91f, in which the gene encodes "Met Leu . . . " instead of"Met-Val-Leu", was likewise constructed from pGEM-FX-A, but with adifferent synthetic replacement oligonucleotide, missing the Val codon.

Plasmid pSGE 1.1 E4

This plasmid (also known as SGE1.1) is depicted in FIG. 1. PlasmidpDLIV-8A may be converted to SGE1.1E4 by the following protocol.

The expression plasmid pDLIV-8a contains the dialpha coding sequences,in which the alpha globins are linked by a single glycine codon, and thedes-val beta globin genes, under the control of a single Ptac promoter.The plasmid encodes ampicillin resistance, does not have a functionaltetracycline resistance gene, and is Rop+ and Lac. Oligonucleotidedirected site specific mutagenesis using a commercially available kitsuch as DoubleTake™ (Stratagene, Inc.) can be used to insert thePresbyterian mutation into the beta globin sequence.

    ______________________________________                                        AAC                    AAA                                                    TTG             →→                                                                     TTT                                                    asn.sup.108            lys                                                    ______________________________________                                    

The final expression plasmid, SGE 1.1E4 (amp R, tet R, Rop-, Lac+) isthen constructed by insertion of both a functional tetracyclineresistance gene and the lacl gene which encodes the lac repressorprotein that inhibits the Ptac promoter until induction with an inducingagent. These modifications are described below.

The initial modification to the plasmid is the insertion of the lacIgene. This gene can be synthesized by polymerase chain reaction (PCR)amplification, according to the manufacturer's protocol (Perkin ElmerCetus, Norwalk, Conn.) using the F episome of E. coli strain JM109(FtraD36, proAB, lacI¶ΔM15)as a substrate. The followingoligonucleotides can be used as primers:

Forward:

    5' GCGGCCGCGGAAGAGTCAATTCAGGAGGGTG 3 '                     (SEQ ID NO:12)

Reverse:

    5' GCGGCCGTCACTGCCCGCTTTCCAGTCGGGAA 3 '                    (SEQ ID NO:13)

The primers contain, at their 5' ends, sequences which encode for NotIrestriction enzyme sites. The product of the PCR amplification reactioncan be blunt ended and cloned into the PuvII site of the expressionplasmid. The PuvII site is not reconstructed during this cloning step,so digestion with PuvII following ligation will linearize plasmids whichdo not incorporate the lacI sequence. Linearized plasmids will nottransform E. coli. Because the primers are complementary only to thetranslated portion of the lacI gene, this fragment does not contain itsown promoter. Note that inducibility or expression of hemoglobin isdependent on the orientation of the lacI gene, thus orientation shouldbe checked after insertion of the lacI gene. The correct orientation hasa smaller EcoR5 fragment than the incorrect orientation. Moreover,insertion of the lacI repressor gene into the PuvII restriction siteinactivates the rop gene product and results in increased plasmid copynumber.

The final modification to the plasmid is restoration of a functionaltetracycline resistance gene. This can be accomplished by digestion ofcommercially available pBR322 with EcoRI followed by insertion vialigation of a synthetic DNA linker containing the 5' and 3' endscomplementary to the EcoRI overhangs and an internal BamHI site. TheBamHI fragment from this modified pBR322 vector containing the 5' codingsequence of the tetracycline resistance gene is purified by agarose gelelectrophoresis, then inserted into the BamHI site of the modified pDLIV-8a plasmid. Only one orientation of the tet^(R) fragment results intetracycline resistance; strains can be screened for the properorientation by growth on the appropriate medium. Insertion of thetet^(R) fragment into the modified vector restores tetracyclineresistance and produces SGE1.1E4.

Plasmid pGEM di-alpha.

The di-alpha gene-bearing SmaI/PstI fragment of SGE 1.1 E4 was ligatedwith SmaI/Psti-cut pGEM 1 to form pGEM di-alpha.

1.1 Subcloning of the α gene into phagescript

The desfxα pGem (pDLII-83a) vector was cut with EcoR1 and Pst1endonucleases and ligated into EcoR1/Pst1 digested phagescript (obtainedfrom Stratagene). E. coil strain DH5α was transformed with the ligationmixture and cells were plated on 2×YT plates overlaid with 3 ml top agarcontaining 10 μl 100 mM IPTG, 25 μl 2% X-Gal in DMSO and 150 μl XL-1cells (Stratagene). Clear plaques were picked and grown at 37° C. in2×YT containing XL-1 cells. Double stranded DNA was isolated from thecultures and checked for the presence of the 500 bp α gene byrestriction analysis and agarose gel electrophoresis. Single strandedDNA was isolated from one of the desfxα phagescript transformants (namedf191). The single stranded DNA was sequenced to confirm the presence ofthe desfxα gene in the phagescript.

1.2 Mutagenic Oligonucleotides

Three mutagenic oligonucleotides were used in three separate mutagenicreactions. The sequences of the oligonucleotides were as follows (mutantcodon is underlined):

Nigeria mutation: αS81C

    5' CCG AAC GCG TTG TGC GCT CTG TCT GAT 3'                   SEQ ID NO:14!

αD75C

    5' GGT GCT CAC GTT GAT TGC ATG CCG AAC GCG 3'               SEQ ID NO:15!

αA71C

    5' CTG ACC AAC GCT GTT TGC CAC GTT GAT GAT 3'               SEQ ID NO:16!

1.3 Kinase reaction conditions for mutagenic oligonucleotides A71C, D75Cand S81C.

1 μl oligonucleotide (approx. 300 pmol)

2 μl 10× kinase buffer containing 10 mM ATP

0.5 μl T4 polynucleotide kinase (10 U/μl, New England Biolabs)

15.5 μl H₂ O

1 μl 10 mM spermidine

Reactions were incubated for 1 hr. at 37° C., then 80 μl H₂ O was addedand the reaction was terminated by heat inactivation at 65° C. for 10min.

1.4 Mutagenesis Reaction

1 μl fw 191 ss DNA (0.5 pmol)

3 μl kinased oligonucleotide (either A71C, D75C or S81C approx. 45 pmol)

2 μl 10× annealing buffer (Promega)

14 μl H₂ O

The no primer control contained:

1 μl fw 191 ss DNA

2 μl 10× annealing buffer

17μl H₂ O

Reactions were heated to 65° C., cooled slowly to 35°C. (approx. 70min), and put on ice for 5 min. The following reagents were added andthe reactions were incubated at 37° C. for 90 min.

3 μl 10× synthesis buffer (Promega)

1 μl T4 gene 32 protein (0.5 μg/μl, Biorad)

1 μl T4 DNA polymerase (3 U/μl, NEB)

0.5 μl T4 DNA ligase (10 U/μl, NEB)

5 μl H₂ O

200 μl 71-18 mut S competent cells (made according to Promega AlteredSites procedure) were transformed with 10 μl of each mutagenesisreaction, put on ice for 30 min and heat shocked for 2 min at 42° C. Thetransformation mixture was added to 3 ml 2×YT media and grown at 37° C.(with shaking) for 5.5 hr. After incubation, 1 ml of each of the threecultures was removed, centrifuged and 800 μl was stored in a fresh tubeat 4° C. as the stock solution of mutant phage.

1.5 Screening for mutants of D75C

100 μl of a 10⁻⁵ dilution of the D75C mutant phage stock was plated on153 mm 2×YT plates overlain with top agar containing 0.5 ml XL-1 cells.Plates were incubated at 37° C. for approx. 5 hrs. Duplicatenitrocellulose filters were lifted off each plate and the plaques werelysed in 6 ml 0.5M NaOH/1.5M NaCl, neutralized in 10 ml 1M Tris-HCl pH8.0/1.5M NaCl and washed in 500 ml 6×SSC. The filters were air dried andbaked at 75° C. for 45 min. The filters were then boiled briefly in 1%SDS prior to prehybridization. Filters were prehybridized in 20 mlsolution for 4 hr at 68° C. The prehybridization on solution was asfollows:

5×SSC (20× SSC prepared according to recipe in Maniatis).

0.1% (w/v) N-lauroylsarcosine

0.02% (w/v) SDS

0.5% blocking reagent (Genius Kit, Boehringer Mannheim)

The D75C oligonucleotide was labelled with .sub.π.sup. 32! ATP follows:

1 μl oligonucleotide (80 pmol)

10 μl 10× kinase buffer

1 μl .sub.π.sup. 32! P ATP (10 μC/μl. Specific activity >3000 Ci/mmol).

87 μl H₂ O

1 μl kinase (10 U/μl, NEB)

The reaction was incubated for 5 hrs. at 37° C. Unincorporated ATP wasremoved by centrifugation through a Biospin 30 column (Biorad). Theentire probe (17,000 cpm/μl) was added to the prehybridization mixtureand the filters were hybridized overnight at 46° C. along with a noprimer control filter. The following day, filters were washed for 10min. at room temperature (RT) in 6×SSC and exposed overnight at -70° C.on Kodak X-Omat film. Filters were washed in 6×SSC at 57° C. for 10 min,dried and exposed overnight, then washed in 6×SSC/0.1% SDS at 67° C. for10 min, and dried and re-exposed overnight. The final was washed in6×SSC/0.1% SDS at 70° C. for 10 min and the filters were again dried andexposed overnight.

Ten plaques were picked which hybridized differentially to the mutantoligonucleotide (compared to the no primer control plaques). The plaqueswere placed in 5 ml 2×YT media containing 0.25 ml XL-1 cells. Thecultures were incubated with shaking at 37° C. for 7.5 hr. 1 ml of eachculture was removed, centrifuged 5 min, placed in a fresh tube andstored at 4° C. for subsequent sequencing and plaque purification.

1.6 Screening for mutants of A71C and S81C

1 μl of a 10⁻³ dilution of the A71C stock phage mutagenesis reaction and20 μl of a 10³¹ 5 dilution of the S81C mutagenesis reaction were platedon four separate 82 mm 2×YT/tet(10 mg/ml) plates overlaid with 3 ml topagar and 100 μl XL-1 cells. A no primer control was also plated asabove. The plates were incubated at 37° C. for 5 hr; plaques were liftedfrom each plate onto nitrocellulose filters and the filters driedovernight at room temperature. The following day, the plaques were lysedwith 0.5M NaOH/1.5M NaCl for 3 min, neutralized in 1M Tris-HCl pH7.0/1.5M NaCl for 3 min and washed in 6×SSC for 5 min. Filters were airdried then baked at 75° C. for 1 hr. The filters were boiled briefly in1.5% SDS prior to prehybridization at 60° C. for 6 hr. in 10 mlprehybridization solution as described above.

1.7 Labelling of A71C and S81C oligonucleotides using digoxigenin

(All reagents supplied by Boehringer Mannheim)

2 μl A71C (100 pmol) or 1 μl S81C (110 pmol)

10 μl terminal transferase buffer

5 μl 25 mM CoCl₂

1 μl 1 mM dUTP-digoxigenin

30 μl 04 31 μl H₂ O (A71C and S81C reactions respectively)

1 μl terminal transferase (25 U/μl)

Reactions were incubated at 37° C. for 3 hr. followed by 6 hr. at RT.Digoxigenin-labelled A71C and S81C probes (20 μl) were added to theappropriate filters in 10 ml prehybridization solution along with a noprimer control filter. The filters were hybridized overnight at 47° C.

1.8 Filter Washes and Development

All filters were initially washed in 6×SSC/0.1% SDS for 15 min at 30°C., then for 15 min at 42° C.

Each of the four filters which had been probed with either the labelledA71C or S81C oligonucleotides were then separated and washed atincreasingly higher temperatures along with a no primer control filter.One each of the A71C and S81C filters were placed in plastic bagscontaining 10 ml of 6×SSC/0.1% SDS and washed for 10 min at one of fourtemperatures, i.e., 50° C., 60° C. or 65° C. After the high temperaturewashes, each set of filters were developed according to the Genius Kitprotocol.

Initially, bags containing the filters were filled with 10 ml of 100 mMTris-HCl, pH 7.5/150 mM NaCl (buffer A) and incubated for 15 min. Thebuffer was removed and replaced with 10 ml buffer A containing 0.5%blocking reagent and incubated a further 15 min at RT without shaking.Anti-digoxigenin antibody (2 μl) was added directly to each bag andincubated with for 30 min at RT. The filters were then removed fromtheir respective bags and washed altogether in 100 ml buffer A/0.05%blocking reagent for 15 min at RT, followed by a 15 min wash in buffer Aalone at RT. The final wash was 100 ml 100 mM Tris-HCl, pH 9.5/100 mMNaCl/50 mM MgCl₂ (buffer B) for 5 min at RT. Each set of filters from agiven temperature was placed in a separate bag along with 5 ml of colordevelopment solution (5 ml buffer B containing 22.5 μl 75 mg/ml NBT/15μl 50 mg/ml X-phosphate). The filters were incubated for 30 min in thedark at RT. After 30 min, the filters were removed from the developmentsolution, washed for 5 min in 100 ml 10×TE and 5 min in 100 ml 7× TE,both at RT. Filters were dried at RT.

Using the results from the Genius Kit screening procedure, 10 plaqueswhich differentially hybridized to the labelled oligonucleotides A71C orS81C were picked and placed in 3 ml 2×YT media containing 0.25 ml XL-1cells and incubated for 7.5 hr. at 37° C. with shaking. 1 ml of eachculture was removed, centrifuged 5 min, placed in a fresh tube andstored at 4° C. for subsequent sequencing and plaque purification.

1.9 Confirmation of mutations by sequencing

Single stranded DNA was isolated from 800 μl mutant phage stocksupernatant and sequenced using the Sequenase kit (USB) with the α 179oligonucleotide as the primer. The α 179 oligonucleotide is an 18-merhomolog cys to a region about 100 bps upstream of the mutation site.Sequencing confirmed the presence of the αA71C, αD75C and α S81Cmutations.

Phage stock was plaque purified by plating 10 μl of 10⁻⁸ and 10⁻¹⁰dilutions on 2×YT/tet (10 mg/ml) plates overlaid with 3 ml top agarcontaining 200 μl XL-1 cells. After 7 hrs incubation at 37° C., a singleisolated plaque from each mutant plate was picked and used to inoculate90 ml 2×YT/tet (10 mg/ml) media containing 10 ml XL-1 cells. Cultureswere grown overnight at 37° C. with shaking. 1 ml of each mutant phageculture was removed, centrifuged and the supernatant was frozen at -80°C. as the respective purified mutant phage stock. Double stranded DNAwas prepared from the remaining culture for use in the subsequentsubcloning steps into the final expression vector 1.1E4.

1.10 Subcloning of the α cys mutants into 1.1E4

Construction of the di-α gone with each of the three cysteine mutationsin either the N-terminal or C-terminal domain of the di-α proteinrequired three subcloning steps:

1) Transfer of the cys mutant α gene from phagescript as an Eag1-Pst1fragment into the Eag1-Pst1 digested desval α pGem vector (pDL II-91f).This step provided the mutant α gene with the correct 5' terminus.

2) A mutant di-α gene with each of the cys mutations in the 3' gene wasconstructed by inserting the Eag1 DNA fragment from di-α pGem (pGemdi-alpha) into the Eag1 site of the relevant cys mutant desval α pGemplasmid. The mutant diα gene with the cys mutation in the 5' α gene wasconstructed by inserting the BstB1 DNA fragment from diα pGem into theBstB1 site of the cys mutant desval α pGem plasmid.

3) Finally each of the mutant diα genes were cloned into the 1.1E4expression vector as a Sma1-Pst1 fragment.

Transformations into DH5α at each step in the subcloning procedure werecarried out as described in the methods of subcloning of the β G83Cmutation into 1.1E4 (see below). The presence of the relevant cysmutation in the correct α gene was confirmed by sequencing at each stagein the subcloning procedure. Each of the diα cys mutants in 1.1E4 weretransformed into E. coli strain 127, grown in TB complete media andinduced with IPTG. Expression of the diα and β proteins was confirmed bySDS-PAGE and Western blot analysis.

Example 2

Protocol for the oxidation of two SGE1.1 monocys's to form apseudo-octamer

The hemoglobin mutants of interest were expressed in E. coli grown instandard fermentation broth, after induction with IPTG. The cells werepelleted, resuspended in 3 mols of 40 mM Tris-base, 2 mM benzamidine/gmcell paste, lysed by two passes through a MICROFLUIDIZER (Microfluidics,Inc.). The lysate was centrifuged to remove cell debris, and thesupernatant was collected. The tetrameric hemoglobin was purified fromthe supernatant using the methodology described in Mathews, et al.(Methods in Enzymology, in press). Briefly, the supernatant is dilutedwith 5 mM Tris, pH 7.0 until the conductivity reaches 200 μmhos. Thesolution is then passed through a flow-through bed of Q SEPHAROSEequilibrated with 20 mM Tris, pH 7.0. The flow through is collected, thepH of the solution is brought up to 8.0 using concentrated NaOH, andthen loaded onto a second Q SEPHAROSE column equilibrated in 20 mM Tris,pH 8.0. The hemoglobin captured on the column is washed with 2 columnvolumes of 20 mM Tris, pH 8.0, and eluted with 20 mM Tris, pH 7.0.Fractions are collected and pooled on the basis of absorbance ratios(A₅₇₅ /A₅₄₀ >1.03). The solution is then buffer exchanged using aMINITAN (Millipore Corp.) into 14 mM sodium phosphate, pH 8-8.5/150 mMapproximately 100 mg/ml. Note that at this point in the purification,some of the mutant hemoglobin tetramers have already formed significantlevels of octamer (>20%) by simple air oxidation of the cysteinesengineered into the molecules. However, to drive oxidation of theremaining cysteine thiols to disulfides, the solution is incubated underair or oxygen for approximately 48 hours at 4° C.

After incubation, the octamers are separated from any unreacted monomerand any higher order polymers using SEPHACRYL S-200 equilibrated in 14mM sodium phosphate, pH 8-8.5/150 mM NaCl at a linear flow rate of 60cm/hr. Fractions are collected and poolings are verified using ananalytical grade SUPEROSE 12 column.

For those hemoglobin mutants where a cysteine replacement is located inclose proximity to a negative charge, or for which disulfide formationis otherwise incomplete, the above procedure is preferably modified toenhance disulfide formation. After concentration to 50 mg/ml using theprocedure described above, the hemoglobin solution is converted tocarbon monoxy hemoglobin by gentle bubbling with CO. Five Cu++/heme areadded to the solution using 100 mM CuCl₂, and the hemoglobin isincubated for 5 minutes on ice, under CO. The reaction is quenched bythe addition of a five-fold molar excess (with respect to copper) ofNa-EDTA. The resulting octamers are then purified as above.

Example 3

Hypothetical protocol for the construction of hemoglobin moleculesstabilized against dimer formation by fusion across the alpha 1-beta 2or alpha 2-beta 1 dimer interface region

The currently employed inter-dimer di-alpha fusion between the Cterminus of one alpha subunit and the N terminus of the adjacent alphasubunit, represents a successful protein engineering approach tostabilizing hemoglobin against dimer formation. In this case, use wasmade of the fortunate juxtaposition of the two termini which originatefrom different dimers. One might also make a di-beta polypeptide, as hasbeen described, or a hemoglobin with both di-alpha and di-betapolypeptide, as has been described, or a hemoglobin with both di-alphaand di-beta linked subunits. Alternatively, one can envision other typesof fusion in which the alpha subunit of one alpha/beta dimer is fused tothe beta subunit of the other dimer (FIG. 6). In this, two individual,linked polypeptides would dimerize to form the psuedo-tetramerichemoglobin. This approach is based on the fact that dimerizationinvolves specific, identical pairs of subunits, generally referred to asα1β1 and α2β2.

As an example of this alternative fusion approach, the alpha subunit Cterminal residue (Arg 141) of dimer 1 could be fused, either directly orwith an intervening fusion sequence, to the N-terminal amino acid of thebeta subunit C helix (Tyr 35) of dimer 2. This would create a new Cterminal residue at the end of the beta B helix (Val 34) and would leavea "free" piece of polypeptide comprised of the beta A and B helices(residues 1 to 34 inclusive). These alterations would give rise to aprotein comprised of alpha subunit helices A through H fused to betasubunit helices C through H. The polypeptide composed of the betasubunit A and B helices would be covalently attached to the protein byintroducing a new helix into the molecule. The helix would be designedto span the distance between the beta C terminus (His 146) and theoriginal beta N terminus of helix A (Val 1). Following these changes,the sequence of helices from the N to C terminus of the new proteinwould be (alpha) A-B-C-E-F'-F-G-H-(beta)-C-D-E-F'-F-G-H-NEW-A-B. Theactual arrangement of the fusion regions would require careful design sothat new regions of structure did not extend into the dimer-dimerinterface region which is critical to cooperativity. Introduction ofamino acids containing basic or acidic residues into the molecule atcertain positions could allow some restoration of functionally importantsalt bridges and hydrogen bonds which could be lost as a result ofmanipulating the normal N and C termini. The above approach could alsoextend to the production of the entire hemoglobin molecule or individualdimers as single polypeptide chains, although in the latter case thiswould not be expected to offer stabilization against dimer formation.

For the purpose of providing the potential for disulfide bond formation,a cysteine may be introduced into either the α or β globin domain of theα₁ β₂ pseudodimer.

Reference Example A

Reconstitution of Recombinant Alpha-Globin and Recombinant Beta-Globinwith Heme and Chemical Reduction to Yield Artificial Hemoglobin

Conventional methods of preparing artificial hemoglobin are exemplifiedby the following procedure.

The lyophilized recombinant alpha and beta-globins (100 mg each) wereindividually dissolved in 8M urea/50 mM Tris-C1, pH 8.01/1 mM EDTA/1 mMDTT, diluted to a concentration of 5 mg/ml and incubated at roomtemperature for 3-4 hours. The alpha-globin was then diluted to 0.3mg/ml with chilled 20 mM K₂ HPO₄, pH 5.7/1 mM EDTA/1 mM DTT. Hemin (25mg) was dissolved in 2.4 mg 0.1M KOH, diluted with an equal volume of 1MKCN; this solution was then made 0.1 mg/ml in hemin and 20 mM K₂ HPO₄,pH 6.7 with stock phosphate buffer. Hemin from this solution was addedat a 2.8 molar excess to the chilled alpha-globin; and equal molaramount of beta-globin was added and the solution was dialyzed at 4° C.overnight against 0.1M K₂ HPO₄, pH 7.6/1 mM EDTA/1 mM KCN. Theartificial Hb solution was concentrated by ultra-filtration using aPM-10 membrane (Amicon) and transferred into a 200 ml screw-top testtube with a rubber septum. The hemoglobin solution was deoxygenated byevacuation and flushing with N₂, and then the solution was saturatedwith CO. 100 mM sodium dithionite solution was prepared anaerobically ina 20 ml screw-top test tube with rubber septum. 4.5 equivalents ofdithionite were added to the Hb solution with a syringe, and the mixtureincubated on ice for 15 min. The Hb solution was gel-filtered against 10mM Na phosphate buffer pH 6.0 on a 4×40 cm SEPHADEX G-25 (fine) column.The colored solution was then applied to a 2×10 cm-52 (Whatman) columnequilibrated with the same buffer and the chromatography was developedwith a linear gradient of 500 ml 10 mM Na phosphate buffer pH 6.0 and500 ml of 70 mM sodium phosphate buffer pH 6.9. CO was removed from Hbby photolysis under a stream of oxygen. Artificial Hgb prepared this wayis isolated in only about 25% yield from the fusion peptides but showsnative oxygen binding properties.

Reference Example B

P₅₀ Determination

Our preferred method of measuring P50 of purified hemoglobin solutionsfor the purpose of the appended claims is as follows:

Hemoglobin-oxygen equilibrium curves are measured using a HEMOX ANALYZER(TCS Medical Products, Southampton, Pa.) at either 25° C. or 37° C.+0.1°C. in 50 mM HEPES buffer/0.1M NaCl, pH 7.4. Oxygen equilibrium curvesare measured by N₂ deoxygenation of an oxyhemoglobin solution that hasbeen previously equilibrated with water-saturated O₂ (for samples with aP50>10 torr) or with water-saturated compressed air. Absorbance readingsat 568 and 558 nm are measured throughout the run for determination ofpercent oxyhemoglobin in the sample. Percent oxyhemoglobin is directlyproportional to log A₅₅₈ /log A₅₆₈ and is independent of path length.Both the absorbances and the oxygen pressure are sampled by aprogrammable-gain 12-bit analog-to-digital converter (Labmaster PGH,Scientific Solutions, Solon, Ohio) under computer control. The oxygenequilibrium curve is subjected to a low pass digital filter. P₅₀ values(partial pressure of O₂ required for 50% saturation of oxygen bindingsites) and Hill coefficients (^(n) max) are calculated from thedigitally filtered data by using software developed in our laboratory.The Hill coefficients are determined as the maximum slope of thefunctions dlog y/(1-y)!/dlog p, where y is % O₂ saturation and p ispartial pressure of O₂.

P₅₀ may also be measured under other conditions, but it should be notedthat many environmental factors affect hemoglobin's oxygen affinity. Theeffect of pH, CO₂ inorganic unions, organic phosphates and temperatureon P₅₀ are discussed in Bunn and Forget, Hemoglobin: Molecular, Geneticand Clinical Aspects 37-47, 95-98 (W. B. Saunders Co., 1986).

Since many determinations of whole blood oxygen binding curves are madeunder standard physiologic conditions (37° C., pH, 7.4, pCO₂ 40 mm Hg),it may be necessary to adjust literature figures. In this context, itshould be noted that a 10° C. increase results in nearly a two-foldincrease in P₅₀, while the dependence of P₅₀ on pH is approximatelygiven as delta log P₅₀ /delta pH=-0.5.

Comparing P₅₀ values of purified Hb preparations to P₅₀ values of wholeblood can be problematic. Whole blood or isolated RBC's contain manycomponents that naturally modulate the shape of the hemoglobin-oxygenbinding curve. The RBC encapsulates Hgb in the presence of a highconcentration of the effector molecule 2,3-DPG; a molecule that causesHgb to have a markedly lower affinity for O₂. Other intra-erythrocytecomponents also affect the shape of the binding curve: ATP, Cl-CO₂, H+,orthophosphate, methemoglobin and carboxyhemoglobin. The levels of thesesubstances may vary with age, sex and condition. These substances arenot normally present in purified Hgb solutions and thus, the P₅₀ valueof purified Hgb is lower than that found in whole blood. One veryimportant modulator of Hgb-oxygen affinity is C1- ion. C1 ion is foundoutside the erythrocyte in the blood serum at a physiologicconcentration of approximately 0.15M. Since C1- causes a lower O₂affinity, a Hgb solution with a P₅₀ measured in vitro may well have muchlower O₂ affinity if infused into the blood stream. Another problem withmeasuring O₂ binding of whole blood is that RBCs are quite fragile andin the process of manipulating the erythrocyte into the instrument usedto measure the O₂ binding it is inevitable that at least a smallpercentage of the RBCs will lyse. Lysed RBCs leak Hgb into thesurrounding media away from 2,3-DPG; hence, since free Hgb has a higheraffinity than intraerythrocyte Hgb, lysed RBCs will have a higher O₂affinity and can cause a falsely low P₅₀ value for whole blood P50determinations. It is widely accepted that under physiologic conditionswhile blood has a P₅₀ value of 26-28 mm Hg. When Hgb is isolated fromwhole blood, however, the measured P₅₀ is on the order of 1-10 mm Hgdepending on the investigator's experimental conditions. For thesereasons it is most accurate to measure Hgb-oxygen equilibria withpurified Hgb molecules under strict conditions of buffer, pH and saltconcentrations. Unfortunately, there are no accepted "standards" for allinvestigators to measure Hgb oxygen binding for in vitro systems.

Still, as many mutant hemoglobins are first identified in patient'swhole blood, one would like to be able to compare the relativeaffinities of native and mutant Hgb for O₂, between whole blood andpurified Hgb preparations. An example of this is Hgb Chico (beta lys⁶⁶-thr). If one examined only the P₅₀ value of the purified mutant Hgb(10.1 mmHg) one would note that Hgb has a P₅₀ value less than that fornormal whole blood (27.2 mmHg). Still, when that hemoglobin is measuredin RBCs under physiologic conditions it is apparent that it does have ahigher P₅₀ than normal whole blood (38 mmHg). One cannot predict thedegree that the P₅₀ value will change going from whole blood Chico to apurified Hgb Chico if it were infused into the bloodstream as a bloodsubstitute. One can conclude however, that the P₅₀ will be higher thanit is in pure form, and that by reacting the mutant Hgb with organicphosphates that P₅₀ will be even higher.

                  TABLE 1                                                         ______________________________________                                        PRIMARY STRUCTURE OF HUMAN GLOBIN SUBUNITS                                    Helix α Zeta   Helix*                                                                              β                                                                              δ                                                                             Gamma  ε                       ______________________________________                                        NA1   1 Val   Ser    NA1   1 Val Val   Gly    Val                                                  NA2   2 His His   His    His                             NA2   2 Leu   Leu    NA3   3 Leu Leu   Phe    Phe                             A1    3 Ser   Thr    A1    4 Thr Thr   Thr    Thr                             A2    4 Pro   Lys    A2    5 Pro Pro   Glu    Ala                             A3    5 Ala   Thr    A3    6 Glu Glu   Glu    Glu                             A4    6 Asp   Glu    A4    7 Glu Glu   Asp    Glu                             A5    7 Lys   Arg    A5    8 Lys Lys   Lys    Lys                             A6    8 Thr   Thr    A6    9 Ser Thr   Ala    Ala                             A7    9 Asn   Ile    A7    10 Ala                                                                              Ala   Thr    Ala                             A8    10 Val  Ile    A8    11 Val                                                                              Val   Ile    Val                             A9    11 Lys  Val    A9    12 Thr                                                                              Asn   Thr    Thr                             A10   12 Ala  Ser    A10   13 Ala                                                                              Ala   Ser    Ser                             A11   13 Ala  Met    A11   14 Leu                                                                              Leu   Leu    Leu                             A12   14 Trp  Trp    A12   15 Trp                                                                              Trp   Trp    Trp                             A13   15 Gly  Ala    A13   16 Gly                                                                              Gly   Gly    Ser                             A14   16 Lys  Lys    A14   17 Lys                                                                              Lys   Lys    Lys                             A15   17 Val  Ile    A15   18 Val                                                                              Val   Val    Met                             A16   18 Gly  Ser                                                             AB1   19 Ala  Thr                                                             B1    20 His  Gln    B1    19 Asn                                                                              Asn   Asn    Asn                             B2    21 Ala  Ala    B2    20 Val                                                                              Val   Val    Val                             B3    22 Gly  Asp    B3    21 Asp                                                                              Asp   Glu    Glu                             B4    23 Glu  Thr    B4    22 Glu                                                                              Ala   Asp    Glu                             B5    24 Tyr  Ile    B5    23 Val                                                                              Val   Ala    Ala                             B6    25 Gly  Gly    B6    24 Gly                                                                              Gly   Gly    Gly                             B7    26 Ala  Thr    B7    25 Gly                                                                              Gly   Gly    Gly                             B8    27 Glu  Glu    B8    26 Glu                                                                              Glu   Glu    Glu                             B9    28 Ala  Thr    B9    27 Ala                                                                              Ala   Thr    Ala                             B10   29 Leu  Leu    B10   28 Leu                                                                              Leu   Leu    Leu                             B11   30 Glu  Glu    B11   29 Gly                                                                              Gly   Gly    Gly                             B12   31 Arg  Arg    B12   30 Arg                                                                              Arg   Arg    Arg                             B13   32 Met  Leu    B13   31 Leu                                                                              Leu   Leu    Leu                             B14   33 Phe  Phe    B14   32 Leu                                                                              Leu   Leu    Leu                             B15   34 Leu  Leu    B15   33 Val                                                                              Val   Val    Val                             B16   35 Ser  Ser    B16   34 Val                                                                              Val   Val    Val                             C1    36 Phe  His    C1    35 Tyr                                                                              Tyr   Tyr    Tyr                             C2    37 Pro  Pro    C2    36 Pro                                                                              Pro   Pro    Pro                             C3    38 Thr  Gln    C3    37 Trp                                                                              Trp   Trp    Trp                             C4    39 Thr  Thr    C4    38 Thr                                                                              Thr   Thr    Thr                             C5    40 Lys  Lys    C5    39 Gln                                                                              Gln   Gln    Gln                             C6    41 Thr  Thr    C6    40 Arg                                                                              Arg   Arg    Arg                             C7    42 Tyr  Tyr    C7    41 Phe                                                                              Phe   Phe    Phe                             CE1   43 Phe  Phe    CD1   42 Phe                                                                              Phe   Phe    Phe                             CE2   44 Pro  Pro    CD2   43 Glu                                                                              Glu   Asp    Asp                             CE3   45 His  His    CD3   44 Ser                                                                              Ser   Ser    Ser                             CE4   46 Phe  Phe    CD4   45 Phe                                                                              Phe   Phe    Phe                                                  CD5   46 Gly                                                                              Gly   Gly    Gly                             CE5   47 Asp  Asp    CD6   47 Asp                                                                              Asp   Asn    Asn                             CE6   48 Leu  Leu    CD7   48 Leu                                                                              Leu   Leu    Leu                             CE7   49 Ser  His    CD8   49 Ser                                                                              Ser   Ser    Ser                             CE8   50 His  Pro    D1    50 Thr                                                                              Ser   Ser    Ser                                                  D2    51 Pro                                                                              Pro   Ala    Pro                                                  D3    52 Asp                                                                              Asp   Ser    Ser                                                  D4    53 Ala                                                                              Ala   Ala    Ala                                                  D5    54 Val                                                                              Val   Ile    Ile                                                  D6    55 Met                                                                              Met   Met    Leu                             CE9   51 Gly  Gly    D7    56 Gly                                                                              Gly   Gly    Gly                             E1    52 Ser  Ser    E1    57 Asn                                                                              Asn   Asn    Asn                             E2    53 Ala  Ala    E2    58 Pro                                                                              Pro   Pro    Pro                             E3    54 Gln  Gln    E3    59 Lys                                                                              Lys   Lys    Lys                             E4    55 Val  Leu    E4    60 Val                                                                              Val   Val    Val                             E5    56 Lys  Arg    E5    61 Lys                                                                              Lys   Lys    Lys                             E6    57 Gly  Ala    E6    62 Ala                                                                              Ala   Ala    Ala                             E7    58 His  His    E7    63 His                                                                              His   His    His                             E8    59 Gly  Gly    E8    64 Gly                                                                              Gly   Gly    Gly                             E9    60 Lys  Ser    E9    65 Lys                                                                              Lys   Lys    Lys                             E10   61 Lys  Lys    E10   66 Lys                                                                              Lys   Lys    Lys                             E11   62 Val  Val    E11   67 Val                                                                              Val   Val    Val                             E12   63 Ala  Val    E12   68 Leu                                                                              Leu   Leu    Leu                             E13   64 Asp  Ala    E13   69 Gly                                                                              Gly   Thr    Thr                             E14   65 Ala  Ala    E14   70 Ala                                                                              Ala   Ser    Ser                             E15   66 Leu  Val    E15   71 Phe                                                                              Phe   Leu    Phe                             E16   66 Thr  Gly    E16   72 Ser                                                                              Ser   Gly    Gly                             E17   68 Asn  Asp    E17   73 Asp                                                                              Asp   Asp    Asp                             E18   69 Ala  Ala    E18   74 Gly                                                                              Gly   Ala    Ala                             E19   70 Val  Val    E19   75 Leu                                                                              Leu   Ile, Thr                                                                             Ile                             E20   71 Ala  Lys    E20   76 Ala                                                                              Ala   Lys    Lys                             EF1   72 His  Ser    EF1   77 His                                                                              His   His    Asn                             EF2   73 Val  Ile    EF2   78 Leu                                                                              Leu   Leu    Met                             EF3   74 Asp  Asp    EF3   79 Asp                                                                              Asp   Asp    Asp                             EF4   75 Asp  Asp    EF4   80 Asn                                                                              Asn   Asp    Asn                             EF5   76 Met  Ile    EF5   81 Leu                                                                              Leu   Leu    Leu                             EF6   77 Pro  Gly    EF6   82 Lys                                                                              Lys   Lys    Lys                             EF7   78 Asn  Gly    EF7   83 Gly                                                                              Giy   Gly    Pro                             EF8   79 Ala  Ala    EF8   84 Thr                                                                              Thr   Thr    Ala                             F1    80 Leu  Leu    F1    85 Phe                                                                              Phe   Phe    Phe                             F2    81 Ser  Ser    F2    86 Ala                                                                              Ser   Ala    Ala                             F3    82 Ala  Lys    F3    87 Thr                                                                              Gln   Gln    Lys                             F4    83 Leu  Leu    F4    88 Leu                                                                              Leu   Leu    Leu                             F5    84 Ser  Ser    F5    89 Ser                                                                              Ser   Ser    Ser                             F6    85 Asp  Glu    F6    90 Glu                                                                              Glu   Glu    Glu                             F7    86 Leu  Leu    F7    91 Leu                                                                              Leu   Leu    Leu                             F8    87 His  His    F8    92 His                                                                              His   His    His                             F9    88 Ala  Ala    F9    93 Cys                                                                              Cys   Cys    Cys                             FG1   89 His  Tyr    FG1   94 Asp                                                                              Asp   Asp    Asp                             FG2   90 Lys  Ile    FG2   95 Lys                                                                              Lys   Lys    Lys                             FG3   91 Leu  Leu    FG3   96 Leu                                                                              Leu   Leu    Leu                             FG4   92 Arg  Arg    FG4   97 His                                                                              His   His    His                             FG5   93 Val  Val    FG5   98 Val                                                                              Val   Val    Val                             G1    94 Asp  Asp    G1    99 Asp                                                                              Asp   Asp    Asp                             G2    95 Pro  Pro    G2    100 Pro                                                                             Pro   Pro    Pro                             G3    96 Val  Val    G3    101 Glu                                                                             Glu   Glu    Glu                             G4    97 Asn  Asn    G4    102 Asn                                                                             Asn   Asn    Asn                             G5    98 Phe  Phe    G5    103 Phe                                                                             Phe   Phe    Phe                             G6    99 Lys  Lys    G6    104 Arg                                                                             Arg   Lys    Lys                             G7    100 Leu Leu    G7    105 Leu                                                                             Leu   Leu    Leu                             G8    101 Leu Leu    G8    106 Leu                                                                             Leu   Leu    Leu                             G9    102 Ser Ser    G9    107 Gly                                                                             Gly   Gly    Gly                             G10   103 His His    G10   108 Asn                                                                             Asn   Asn    Asn                             G11   104 Cys Cys    G11   109 Val                                                                             Val   Val    Val                             G12   105 Leu Leu    G12   110 Leu                                                                             Leu   Leu    Met                             G13   106 Leu Leu    G13   111 Val                                                                             Val   Val    Val                             G14   107 Val Val    G14   112 Cys                                                                             Cys   Thr    Ile                             G15   198 Thr Thr    G15   113 Val                                                                             Val   Val    Ile                             G16   109 Leu Leu    G16   114 Leu                                                                             Leu   Leu    Leu                             G17   110 Ala Ala    G17   115 Ala                                                                             Ala   Ala    Ala                             G18   111 Ala Ala    G18   116 His                                                                             Arg   Ile    Thr                             G19   112 His Arg    G19   117 His                                                                             Asn   His    His                             GH1   113 Leu Phe    GH1   118 Phe                                                                             Phe   Phe    Phe                             GH2   114 Pro Pro    GH2   119 Gly                                                                             Gly   Gly    Gly                             GH3   115 Ala Ala    GH3   120 Lys                                                                             Lys   Lys    Lys                             GH4   116 Glu Asp    GH4   121 Glu                                                                             Glu   Glu    Glu                             GH5   117 Phe Phe    GH5   122 Phe                                                                             Phe   Phe    Phe                             H1    118 Thr Thr    H1    123 Thr                                                                             Thr   Thr    Thr                             H2    119 Pro Ala    H2    124 Pro                                                                             Pro   Pro    Pro                             H3    120 Ala Glu    H3    125 Pro                                                                             Gln   Glu    Glu                             H4    121 Val Ala    H4    126 Val                                                                             Met   Val    Val                             H5    122 His His    H5    127 Gln                                                                             Gln   Gln    Gln                             H6    123 Ala Ala    H6    128 Ala                                                                             Ala   Ala    Ala                             H7    124 Ser Ala    H7    129 Ala                                                                             Ala   Ser    Ala                             H8    125 Leu Trp    H8    130 Tyr                                                                             Tyr   Trp    Trp                             H9    126 Asp Asp    H10   131 Gln                                                                             Gln   Gln    Gln                             10    127 Lys Lys    H10   132 Lys                                                                             Lys   Lys    Lys                             H11   128 Phe Phe    H11   133 Val                                                                             Val   Met    Leu                             H12   129 Leu Leu    H12   134 Val                                                                             Val   Val    Val                             H13   130 Ala Ser    H13   135 Ala                                                                             Ala   Thr    Ser                             H14   131 Ser Val    H14   136 Gly                                                                             Gly   Gly, Ala                                                                             Ala                             H15   132 Val Val    H15   137 Val                                                                             Val   Val    Val                             H16   133 Ser Ser    H16   138 Ala                                                                             Ala   Ala    Ala                             H17   134 Thr Ser    H17   139 Asn                                                                             Asn   Ser    Ile                             H1B   135 Val Val    H18   140 Ala                                                                             Ala   Ala    Ala                             H19   136 Leu Leu    H19   141 Leu                                                                             Leu   Leu    Leu                             H20   137 Thr Thr    H20   142 Ala                                                                             Ala   Ser    Ala                             H21   138 Ser Glu    H21   143 His                                                                             His   Ser    His                             HC1   139 Lys Lys    HC1   144 Lys                                                                             Lys   Arg    Lys                             HC2   140 Tyr Tyr    HC2   145 Tyr                                                                             Tyr   Tyr    Tyr                             HC3   141 Arg Arg    HC3   146 His                                                                             His   His    His                             (SEQ ID (SEQ ID        (SEQ ID                                                                              (SEQ ID                                                                              (SEQ ID                                                                              (SEQ                              NO:17)  NO:18)         NO:19) NO:20) NO:21-)                                                                              ID                                                                     23     NO:                                                                           24)                               ______________________________________                                    

                                      TABLE 2                                     __________________________________________________________________________    NATURAL LOW AFFINITY HEMOGLOBIN MUTANTS                                       __________________________________________________________________________                     P.sub.50 *                                                   Hemoglobin                                                                           Alpha Mutant                                                                            RBC-Free Hgb                                                                         Whole Blood (nl)                                                                      Area of Mutant( )Reference                    __________________________________________________________________________    Hirosaki                                                                             43(CD1) phe-->leu                                                                       n/a            heme   1,2                                    Torino 43(CD1) phe-->val                                                                       n/a            heme   1,3                                    Moabit 86(F7) leu-->arg 30.6 (26.4-29.2)                                                                      heme    4                                     Titusville                                                                           94(G1) asp-->asn                                                                        15.8(4.7)      α.sub.1 β.sub.2                                                            5                                     __________________________________________________________________________                     P.sub.50 (mmHg)                                              Hemoglobin                                                                           Beta Mutant                                                                             Hab (nl)                                                                             Whole Blood (nl)                                                                      Area of Mutant( )Reference                    __________________________________________________________________________    Raleigh                                                                              1 val-->acetyl ala                                                                      4.0(2.2)       DPG site                                                                              6                                     Connecticut                                                                          21(B3) asp-->gly                                                                        5.0(2.2)       B-E helices                                                                           7                                     Moscva 24(B6) gly-->asp                                                                        14.8(12.6)     B-E helices                                                                           8                                     Rothschild                                                                           37(C3) trp-->arg                                                                        3.5(2.0)       α.sub.1 β.sub.2                                                            9                                     Hazebrouck                                                                           38(C4) thr-->pro 36 (27-29)                                                                            α.sub.1 β.sub.2                                                           10                                     Hammersmith                                                                          42(CD1) phe-->ser                                                                       n/a            heme/α.sub.1 β.sub.2                                                      1,11                                   Louisville                                                                           42(CD1) phe-->leu                                                                       24(21)         heme/α.sub.1 β.sub.2                                                      12,13                                  Sendagi                                                                              42(CD1) phe-->val                                                                       3.75(3.05)     heme/α.sub.1 β.sub.2                                                      14                                     Cheverley                                                                            45(CD4) phe-->ser                                                                              38.7 (28.7)                                                                           heme   15                                     Okaloosa                                                                             48(CD7) leu-->arg                                                                       0.95(0.7)                                                                            30(26)  C-D helices                                                                          16                                     Bologna                                                                              61(E5) lys-->met 37.6 (27.0)                                                                           B-E helices                                                                          17                                     Cairo  65(E9) lys-->gln 41(31)  heme   18                                     Chico  66(E10) lys-->thr                                                                       10.1(5.0)                                                                            38.0 (27.2)                                                                           heme   19                                     Bristol                                                                              67(E11) val-->asp                                                                              25.0 (19.0)                                                                           heme   20                                     Seattle                                                                              70(E14) ala-->asp                                                                              43.5 (28.1)                                                                           heme   21,22                                  Vancouver                                                                            73(E17) asp-->tyr                                                                       n/a                   1,23                                   Korle-Bu                                                                             73(E17) asp-->asn                                                                       n/a                   1,24                                   Mobile 73(E17) asp-->val                                                                       n/a                                                          Rahere 82(EF6) lys-->thr                                                                       15.5(11.0)     DPG site                                                                             26                                     Pyrgos 83(EF7) gly-->asp        External                                                                             27                                     Roseau-Pointe                                                                        90(F6) glu-->gly 38(28)  α.sub.1 β.sub.2                                                           28                                     Agenogi                                                                              90(F6) glu-->lys                                                                        9.0(6.8)       α.sub.1 β.sub.2                                                           29                                     Caribbean                                                                            91(F7) leu-->arg                                                                        28.0(21.0)     heme   30                                     Kansas 102(G4) asn-->thr                                                                       28.0(9.0)      α.sub.1 β.sub.2                                                           31                                     Beth Israel                                                                          102(G4) asn-->ser                                                                              88.0 (26.0)                                                                           α.sub.1 β.sub.2                                                           32                                     Saint Mande                                                                          102(G4) asn-->tyr                                                                              52 (28) α.sub.1 β.sub.2                                                           33                                     Richmond                                                                             102(G4) asn-->lys                                                                       n/a            α.sub.1 β.sub.2                                                           1,34                                   Burke  107(G9) gly-->arq                                                                       9.3(7.7)       heme   35                                     Yoshizuka                                                                            108(G10) asn-->asp                                                                      12.9(9.0)      α.sub.1 β.sub.1                                                           36                                     Presbyterian                                                                         108(G10) asn-->lys                                                                      6.3(2.5)       α.sub.1 β.sub.1                                                           37                                     Peterborough                                                                         111(G13) val-->phe                                                                      14.0(9.0)      α.sub.1 β.sub.1                                                           38                                     New York                                                                             113(G15) val-->glu                                                                      n/a            G-helix                                                                              1,39                                   Hope   136(H14) gly-->asp                                                                      n/a            heme   1,40                                   Himeji 140(H18) ala-->asp                                                                      5.8(4.5)                                                     __________________________________________________________________________    *Parenthetical values are that investigator's measured P.sub.50 for           conventional Hgb A in RBC-free or                                             RBC-bound state, as indicated                                                 References for Table 2                                                        1) Wrightstone, R. N. Hemoglobin 1987, 11, 241-308.                           2) Ohba, Y.; Miyaji, T.; Matsuoka, M.; Yokoyama, M.; Numakura, H.;            Nagata, K.; Takebe, Y.; Izumu,                                                Y.; Shibata, S. Biochem. Biophys. Acta 1975, 405, 155-160.                    3) Beretta, A.; Prato, V.; Gallo, E.; Lehmann, H. Nature 1968, 217,           1016-1018.                                                                    4) Knuth, A.; Pribilla, W.; Marti, H. R.; Winterhalter, K. H. Acta            Haematol 1979, 61, 121-124.                                                   5) Schneider, R. G.; Atkins, R. J.; Hosty, T. S.; Tomlin, G.; Casey, R.;      Lehmann, H.; Lorkin, P. A.;                                                   Nagai, K. Biochem. Biophys. Acta 1975, 400, 365-373.                          6) Moo-Penn, W. F.; Bechtel, K.C.; Schmidt, R. M.; Johnson, M. H.; Jue,       D. L.; Schmidt, D. E.; Dunlap,                                                W. M.; Opella, S. J.; Bonaventura, J.; Bonaventura C. Biochemistry 1977,      16, 4872-4879.                                                                7) Moo-Penn, W. F.; McPhedran, P.; Bobrow, S.; Johnson, M. H.; Jue, D.        L.; Olsen, K. W. Amer. J.                                                     Hermtol. 1981, 11, 137-145.                                                   8) Idelson, L. I.; Didkowsky, N. A.; Casey, R.; Lorkin, P. A.; Lehmann,       H. Nature 1974, 249, 768-770.                                                 9) Gacon, G.; Belkhodja, O.; Wajcman, H.; Labie, D. Febs Lett 1977, 82,       243-246.                                                                      10) Blouquit.; Delanoe,-Garin, J.; Lacombe, C.; Arous, N.; Cayre, Y.;         Peduzzi, J.; Braconnier, F.;                                                  Galacteros, F.; Febs Lett. 1984, 172, 155-158.                                11) Dacie, J. V.; Shinton, N. K.; Gaffney, P. J.; Carrell, R. W.;             Lehmann, H. Nature 1967, 216, 663-665.                                        12) Keeling, M. M.; Ogden, L. L.; Wrightstone, R. N.; Wilson, J. B.;          Reynolds, C. A.; Kitchens, J. L.;                                             Huisman, T. H. J. Clin. Invest. 1971, 50, 2395-2402.                          13) Bratu, V.; Larkin, P. A.; Lehmann, H.; Predescu, C. Biochem. Biophys.     Acta. 1971, 251, 1-6.                                                         14) Ogata, K.; Ho, T.; Okazaki, T.: Dan, K.; Nomura, T.; Nozawa, Y.;          Kajita, A. Hemoglobin 1986, 10,                                               469-481.                                                                      15) Yeager, A. M.; Zinkham, W. H.; Jue, D. L.; Winslow, R. M.; Johnson,       M. H.; McGuffey, J. E.;                                                       Moo-Penn, W. F. Ped. Res. 1983, 17, 503-507.                                  16) Charache, S.; Brimhall, B.; Milner, P.; Cobb, L. J. Clin. Invest.         1973, 52, 2858-2864.                                                          17) Marinucci, M.; Giuliani, A.; Maffi, D.; Massa, A.; Giampolo, A.;          Mavilio, F.; Zannotti, M.; Tantori,                                           L. Biochem. Biophys. Acta. 1981, 668, 209-215.                                18) Garel, M. C.; Hasson, W.; Coquelet, M. T.; Goosens, M.; Rosa, J.;         Arous, N. Biochem. Biophys.                                                   Acta. 1976, 420, 97-104.                                                      19) Shih, D. T.; Jones, R. T.; Shih, M. F. C.; Jones, M. B.; Koler, R.        D.; Hemoglobin 1987, 11, 453-464.                                             20) Steadman, J. H.; Yates, A.; Huehns, E. R.; Brit. J Haematol 1970, 18,     435-446.                                                                      21) Stamatoyannopoulos, G.; Parer, J. T.; Finch, C. New Eng. J. Med.          1969, 281, 915-919.                                                           22) Anderson, N. L.; Perutz, M. F.; Stamatoyannopoulos, G. Nature New         Biol. 1973, 243, 275-276.                                                     23) Jones, R. T.; Brimhall, B.; Pootrakul, S.; Gray, G. J. Mol. Evol.         1976, 9, 37-44.                                                               24) Konotey-Ahulu, F. I. D.; Gallo, E.; Lehmann, H.; Ringelhann, B. J.        Med. Genet. 1968, 5, 107-111.                                                 25) Schneider, R. G.; Hosty, T. S.; Tomlin, G.; Atkins, R.; Brimhall, B.;     Jones, R. T. Biochem. Genet.                                                  1975, 13, 411-415.                                                            26) Sugihara, J.; Imamura, T.; Nagafuchi, S.; Bonaventura, J.;                Bonaventura, C.; Cashon, R. J. Clin.                                          Invest. 1985, 76, 1169-1173.                                                  27) Tatsis, B.; Sofroniadou, K.; Stergiopoulas, C. I. Blood 1976, 47,         827-832.                                                                      28) Merault, G.; Keclard, L.; Saint-Martin, C.; Jasmin, K.; Campier, A.;      Delanoe Garin, J.; Arous, N.;                                                 Fortune, R.; Theodore, M.; Seytor, S.; Rosa, J.; Blouquit, Y.;                Galacteros, F. Febs Lett. 1985, 184, 10-13.                                   29) Imai, K.; Morimoto, H.; Kotani, M.; Shibata, S.; Miyaji, T.1              Matsutomo, K. Biochem. Biophys. Acta.                                         1970, 200, 197-202.                                                           30) Ahern, E.; Ahern, V.; Hilton, T.; Serjeant, G. D.; Serjeant, B. E.;       Seakins, M.; Lang, A.; Middleton,                                             A.; Lehmann, H. Febs Lett. 1976, 69, 99-102.                                  31) Bonaventura, J.; Riggs, A.; J. Biol Chem. 1968, 243, 980-991.             32) Nagel, R. L.; Lynfield, J.; Johnson, J.; Landeau, L.; Bookchin; R.        M.; Harris, M. B. N. Eng. J. Med.                                             1976, 295, 125-130.                                                           33) Arous, N.; Braconnier, F.; Thillet, J.; Blouquit, Y.; Galacteros, F.;     Chevrier, M.; Bordahandy, C.;                                                 Rosa, J. Febs Lett. 1981, 126, 114-116.                                       34) Efremov, G. D.; Huisman, T. H. J.; Smith, L. L.; Wilson, J. B.;           Kitchens, J. L.; Wrightston, R. N.;                                           Adams, H. R.; J. Biol. Chem. 1969, 244, 6105-6116.                            35) Turner, J. W.; Jones, R. T.; Brimhall, B.; DuVal, M. C.; Koler, R. D.     Biochem. Genet. 1976, 14,                                                     577-585.                                                                      36) Imamura, T.; Fujita, S.; Ohta, Y.; Hanada, M.; Yanase, T. J. Clin.        Invest. 1969, 48, 2341-2348.                                                  37) Moo-Penn, W. F.; Wolff, J. A.; Simon, G., Vacek, M.; Jue, D. L.;          Johnson, M. H. Febs Lett.1978,                                                92, 53-56.                                                                    38) King, M. A. R.; Willshire, B. G.; Lehmann, H., Marimoto, H. Br. J.        Haem. 1972, 22, 125-134.                                                      39) Ranney, H. M.; Jacobs, A. S.; Nagel, R. L. Nature 1967, 213,              876-878.                                                                      40) Minnich, V.; Hill, R. J.; Khuri, P. D.; Anderson M. E. Blood 1965,        25, 830-838.                                                                  41) Ohba, Y.; Miyaji, T.; Murakami, M.; Kadowaki, S.; Fujita, T.; Oimoni,     M.; Hatanaka, H.; Ishikawa,                                                   K.; Baba, S.; Hitaka, K,; Imai, K. Hemoglobin 1986, 10, 109-126.          

                  TABLE 3                                                         ______________________________________                                        Candidate Non-Naturally Occurring Low                                         Affinity Hemoglobin Mutants                                                   ______________________________________                                                     alpha chain                                                                   46 phe-->thr                                                                  46 phe-->ser                                                                  46 phe-->ala                                                                  58 his-->phe                                                                  58 his-->trp                                                                  61 lys-->thr                                                                  61 lys-->ser                                                                  61 lys-->met                                                                  61 lys-->asn                                                                  62 val-->leu                                                                  62 val-->ile                                                                  62 val-->phe                                                                  62 val-->trp                                                                  65 ala-->asp                                                                  94 asp-->gln                                                                  94 asp-->thr                                                                  94 asp-->ser                                                                  94 asp-->lys                                                                  94 asp-->gly                                                                  94 asp-->arg                                                                  beta chain                                                                    21 asp-->ala                                                                  21 asp-->ser                                                                  45 phe-->ala                                                                  45 phe-->thr                                                                  45 phe-->val                                                                  63 his-->phe                                                                  63 his-->trp                                                                  66 lys-->ser                                                                  66 lys-->asn                                                                  67 val-->phe                                                                  67 val-->trp                                                                  67 val-->ile                                                                  70 ala-->glu                                                                  70 ala-->ser                                                                  70 ala-->thr                                                                  96 leu-->phe                                                                  96 leu-->his                                                                  96 leu-->lys                                                                  98 val-->trp                                                                  98 val-->phe                                                                  102 asn-->asp                                                                 102 asn-->glu                                                                 102 asn-->arg                                                                 102 asn-->his                                                                 102 asn-->gly                                                                 108 asn-->arg                                                                 108 asn-->glu                                                    ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                        HIGH OXYGEN AFFINITY, NATURALLY OCCURRING                                     HEMOGLOBIN MUTANTS                                                            Structure          Name                                                       ______________________________________                                        A.     Alpha Chain Mutants                                                           6 (A4)          Asp->AlaSawara                                                                Asp->AsnDunn                                                                  Asp->ValFerndown                                                              Asp->TyrWoodville                                                             Lys->AsnAlbany-Suma                                           40 (C5)         Lys->GluKariya                                                44 (CE2)        Pro->LeuMilledgeville                                                         Pro->ArgKawachi                                               45 (CE3)        His->ArgFort de France                                        85 (F6)         Asp->AsnG-Norfolk                                             92 (FG4)        Arg->GlnJ-Cape Town                                                           Arg->LeuChesapeake                                            95 (G2)         Pro->LeuG-Georgia                                                             Pro->SerRampa                                                                 Pro->AlaDenmark Hill                                                          Pro->ArgSt. Luke's                                            97 (G4)         Asn->LysDallas                                                126 (H9)        Asp->AsnTarrant                                               141 (HC3)       Arg->HisSuresnes                                                              Arg->SerJ-Cubujuqui                                                           Arg->LeuLegnano                                        B.     Beta Chain Mutants                                                            2 (NA2)         His->ArgDeer Lodge                                                            His->GlnOkayama                                               20 (B2)         Val->MetOlympia                                               23 (B5)         Val->AspStrasbourg                                                            Val->PhePalmerston North                                      34 (B16)        Val->PhePitie-Salpetriere                                     36 (C2)         Pro->ThrLinkoping                                             37 (C3)         Trp->SerHirose                                                40 (C6)         Arg->LysAthens-Ga                                                             Arg->SerAustin                                                51 (D2)         Pro->ArgWillamette                                                            Leu->HisBrisbane                                              79 (EF3)        Asp->Gly G-Hsi-Tsou                                                           Lys->ThrRahere                                                                Lys->MetHelsinki                                              89 (F5)         Ser->AsnCreteil                                                               Ser->ArgVanderbilt                                            94 (FG1)        Asp->HisBarcelona                                                             Asp->AsnBunbury                                               96 (FG3)        Leu->Val Regina                                               97 (FG4)        His->GlnMalmo                                                                 His->LeuWood                                                  99 (G1)         Asp->AsnKempsey                                                               Asp->HisYakima                                                                Asp->AlaRadcliffe                                                             Asp->Tyr Ypsilanti                                                            Asp->GlyHotel-Dieu                                                            Asp->Val Chemilly                                             100 (G2)        Pro->LeuBrigham                                               101 (G3)        Glu->LysBritish Columubia                                                     Glu->GlyAlberta                                                               Glu->AspPotomac                                               103 (G5)        Phe->LeuHeathrow                                              109 (G11)       Val->MetSan Diego                                             121 (GH4)       Glu->GlnD-Los Angeles                                                         Pro->GlnTu Gard                                                               Ala->Procrete                                                 140 (H18)       Ala->ThrSt.-Jacques                                           142 (H20)       Ala->AspOhio                                                  143 (H21)       His->ArgAbruzzo                                                               His->GlnLittle Rock                                                           His->ProSyracuse                                              144 (HC1)       Lys->AsnAndrew-Minneapolis                                    145 (HC2)       Tyr->HisBethesda                                                              Tyr->CysRainier                                                               Tyr->AspFort Gordon                                                           Tyr->TermMcKees Rocks                                         146 (HC3)       His->AspHiroshima                                                             His->ProYork                                                                  His->LeuCowtown                                        ______________________________________                                    

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 35                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1464 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       AATTCGAGCTCGGTACCCGGGCTACATGGAGATTAACTCAATCTAGAGGGTATTAATAAT60                GTATCGCTTAAATAAGGAGGAATAACATATGCTGTCTCCGGCCGATAAAACCAACGTTAA120               AGCTGCTTGGGGTAAAGTTGGCGCGCACGCTGGTGAATACGGTGCTGAAGCTCTCGAGCG180               TATGTTCCTGTCTTTCCCGACCACCAAAACCTACTTCCCGCACTTCGACCTGTCTCACGG240               TTCTGCGCAGGTTAAAGGTCACGGTAAAAAAGTTGCTGATGCTCTGACCAACGCTGTTGC300               TCACGTTGATGATATGCCGAACGCGTTGTCTGCTCTGTCTGATCTGCACGCTCACAAACT360               GCGTGTTGATCCGGTTAACTTCAAACTGCTGTCTCACTGCCTGCTGGTTACTCTGGCTGC420               TCATCTGCCGGCTGAATTTACCCCGGCTGTTCATGCGTCTCTGGATAAATTCCTGGCTTC480               TGTTTCTACCGTTCTGACTTCGAAATACCGTGGTGTTCTGTCTCCGGCCGATAAAACCAA540               CGTTAAAGCTGCTTGGGGTAAAGTTGGCGCGCACGCTGGTGAATACGGTGCTGAAGCTCT600               CGAGCGTATGTTCCTGTCTTTCCCGACCACCAAAACCTACTTCCCGCACTTCGACCTGTC660               TCACGGTTCTGCGCAGGTTAAAGGTCACGGTAAAAAAGTTGCTGATGCTCTGACCAACGC720               TGTTGCTCACGTTGATGATATGCCGAACGCGTTGTCTGCTCTGTCTGATCTGCACGCTCA780               CAAACTGCGTGTTGATCCGGTTAACTTCAAACTGCTGTCTCACTGCCTGCTGGTTACTCT840               GGCTGCTCATCTGCCGGCTGAATTTACCCCGGCTGTTCATGCGTCTCTGGATAAATTCCT900               GGCTTCTGTTTCTACCGTTCTGACTTCGAAATACCGTTAATGACTGCAGCTACATGGAGA960               TTAACTCAATCTAGAGGGTATTAATAATGTATCGCTTAAATAAGGAGGAATAACATATGC1020              ACCTGACTCCGGAAGAAAAATCCGCGGTTACTGCTCTGTGGGGTAAAGTGAACGTTGACG1080              AAGTTGGTGGTGAAGCTCTGGGACGTCTGCTGGTTGTTTACCCGTGGACTCAGCGTTTCT1140              TTGAATCTTTCGGAGATCTGTCTACCCCGGACGCTGTTATGGGTAACCCGAAAGTTAAAG1200              CCCATGGTAAAAAAGTTCTGGGTGCTTTCTCTGACGGTCTGGCTCACCTGGACAACCTGA1260              AAGGTACCTTCGCTACTCTGTCTGAGCTCCACTGCGACAAACTGCACGTTGACCCGGAAA1320              ACTTCCGTCTGCTGGGTAAAGTACTAGTTTGCGTTCTGGCTCACCACTTCGGTAAAGAAT1380              TCACTCCGCCGGTTCAGGCTGCTTACCAGAAAGTTGTTGCTGGTGTTGCTAACGCGCTAG1440              CTCACAAATACCACTAATGAAGCT1464                                                  (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       IleGluGlyArg                                                                  (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       ValHisLeuThrPro                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       SerLysTyrArg                                                                  1                                                                             (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       ValLeuSerPro                                                                  1                                                                             (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       HisLysTyrHis                                                                  1                                                                             (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       ValHisLeuThr                                                                  1                                                                             (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       LysCysAlaGluLeuGluGlyArgLeuGluAlaLeuGluGlyArgLeu                              151015                                                                        GluAlaLeuGluGlyArgLeuGluAlaLeuGluGlyArgLeuGluAla                              202530                                                                        LeuGluGlyLysLeu                                                               35                                                                            (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       GlyGluLeuGluGluLeuLeuLysLysLeuLysGluLeuLeuLysGly                              151015                                                                        (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      GAAGCCAUGG10                                                                  (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      IleGluGlyArg                                                                  1                                                                             (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      GCGGCCGCGGAAGAGTCAATTCAGGAGGGTG31                                             (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      GCCGGCCGTCACTGCCCGCTTTCCAGTCGGGAA33                                           (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      CCGAACGCGTTGTGCGCTCTGTCTGAT27                                                 (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      GGTGCTCACGTTGATTGCATGCCGAACGCG30                                              (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      CTGACCAACGCTGTTTGCCACGTTGATGAT30                                              (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 141 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      ValLeuSerProAlaAspLysThrAsnValLysAlaAlaTrpGlyLys                              151015                                                                        ValGlyAlaHisAlaGlyGluTyrGlyAlaGluAlaLeuGluArgMet                              202530                                                                        PheLeuSerPheProThrThrLysThrTyrPheProHisPheAspLeu                              354045                                                                        SerHisGlySerAlaGlnValLysGlyHisGlyLysLysValAlaAsp                              505560                                                                        AlaLeuThrAsnAlaValAlaHisValAspAspMetProAsnAlaLeu                              65707580                                                                      SerAlaLeuSerAspLeuHisAlaHisLysLeuArgValAspProVal                              859095                                                                        AsnPheLysLeuLeuSerHisCysLeuLeuValThrLeuAlaAlaHis                              100105110                                                                     LeuProAlaGluPheThrProAlaValHisAlaSerLeuAspLysPhe                              115120125                                                                     LeuAlaSerValSerThrValLeuThrSerLysTyrArg                                       130135140                                                                     (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 141 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      SerLeuThrLysThrGluArgThrIleIleValSerMetTrpAlaLys                              151015                                                                        IleSerThrGlnAlaAspThrIleGlyThrGluThrLeuGluArgLeu                              202530                                                                        PheLeuSerHisProGlnThrLysThrTyrPheProHisPheAspLeu                              354045                                                                        HisProGlySerAlaGlnLeuArgAlaHisGlySerLysValValAla                              505560                                                                        AlaValGlyAspAlaValLysSerIleAspAspIleGlyGlyAlaLeu                              65707580                                                                      SerLysLeuSerGluLeuHisAlaTyrIleLeuArgValAspProVal                              859095                                                                        AsnPheLysLeuLeuSerHisCysLeuLeuValThrLeuAlaAlaArg                              100105110                                                                     PheProAlaAspPheThrAlaGluAlaHisAlaAlaTrpAspLysPhe                              115120125                                                                     LeuSerValValSerSerValLeuThrGluLysTyrArg                                       130135140                                                                     (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 146 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      ValHisLeuThrProGluGluLysSerAlaValThrAlaLeuTrpGly                              151015                                                                        LysValAsnValAspGluValGlyGlyGluAlaLeuGlyArgLeuLeu                              202530                                                                        ValValTyrProTrpThrGlnArgPhePheGluSerPheGlyAspLeu                              354045                                                                        SerThrProAspAlaValMetGlyAsnProLysValLysAlaHisGly                              505560                                                                        LysLysValLeuGlyAlaPheSerAspGlyLeuAlaHisLeuAspAsn                              65707580                                                                      LeuLysGlyThrPheAlaThrLeuSerGluLeuHisCysAspLysLeu                              859095                                                                        HisValAspProGluAsnPheArgLeuLeuGlyAsnValLeuValCys                              100105110                                                                     ValLeuAlaHisHisPheGlyLysGluPheThrProProValGlnAla                              115120125                                                                     AlaTyrGlnLysValValAlaGlyValAlaAsnAlaLeuAlaHisLys                              130135140                                                                     TyrHis                                                                        145                                                                           (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 146 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      ValHisLeuThrProGluGluLysThrAlaValAsnAlaLeuTrpGly                              151015                                                                        LysValAsnValAspAlaValGlyGlyGluAlaLeuGlyArgLeuLeu                              202530                                                                        ValValTyrProTrpThrGlnArgPhePheGluSerPheGlyAspLeu                              354045                                                                        SerSerProAspAlaValMetGlyAsnProLysValLysAlaHisGly                              505560                                                                        LysLysValLeuGlyAlaPheSerAspGlyLeuAlaHisLeuAspAsn                              65707580                                                                      LeuLysGlyThrPheSerGlnLeuSerGluLeuHisCysAspLysLeu                              859095                                                                        HisValAspProGluAsnPheArgLeuLeuGlyAsnValLeuValCys                              100105110                                                                     ValLeuAlaArgAsnPheGlyLysGluPheThrProGlnMetGlnAla                              115120125                                                                     AlaTyrGlnLysValValAlaGlyValAlaAsnAlaLeuAlaHisLys                              130135140                                                                     TyrHis                                                                        145                                                                           (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 146 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      GlyHisPheThrGluGluAspLysAlaThrIleThrSerLeuTrpGly                              151015                                                                        LysValAsnValGluAspAlaGlyGlyGluThrLeuGlyArgLeuLeu                              202530                                                                        ValValTyrProTrpThrGlnArgPhePheAspSerPheGlyAsnLeu                              354045                                                                        SerSerAlaSerAlaIleMetGlyAsnProLysValLysAlaHisGly                              505560                                                                        LysLysValLeuThrSerLeuGlyAspAlaIleLysHisLeuAspAsp                              65707580                                                                      LeuLysGlyThrPheAlaGlnLeuSerGluLeuHisCysAspLysLeu                              859095                                                                        HisValAspProGluAsnPheLysLeuLeuGlyAsnValLeuValThr                              100105110                                                                     ValLeuAlaIleHisPheGlyLysGluPheThrProGluValGlnAla                              115120125                                                                     SerTrpGlnLysMetValThrAlaValAlaSerAlaLeuSerSerArg                              130135140                                                                     TyrHis                                                                        145                                                                           (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 146 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      GlyHisPheThrGluGluAspLysAlaThrIleThrSerLeuTrpGly                              151015                                                                        LysValAsnValGluAspAlaGlyGlyGluThrLeuGlyArgLeuLeu                              202530                                                                        ValValTyrProTrpThrGlnArgPhePheAspSerPheGlyAsnLeu                              354045                                                                        SerSerAlaSerAlaIleMetGlyAsnProLysValLysAlaHisGly                              505560                                                                        LysLysValLeuThrSerLeuGlyAspAlaIleLysHisLeuAspAsp                              65707580                                                                      LeuLysGlyThrPheAlaGlnLeuSerGluLeuHisCysAspLysLeu                              859095                                                                        HisValAspProGluAsnPheLysLeuLeuGlyAsnValLeuValThr                              100105110                                                                     ValLeuAlaIleHisPheGlyLysGluPheThrProGluValGlnAla                              115120125                                                                     SerTrpGlnLysMetValThrGlyValAlaSerAlaLeuSerSerArg                              130135140                                                                     TyrHis                                                                        145                                                                           (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 146 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      GlyHisPheThrGluGluAspLysAlaThrIleThrSerLeuTrpGly                              151015                                                                        LysValAsnValGluAspAlaGlyGlyGluThrLeuGlyArgLeuLeu                              202530                                                                        ValValTyrProTrpThrGlnArgPhePheAspSerPheGlyAsnLeu                              354045                                                                        SerSerAlaSerAlaIleMetGlyAsnProLysValLysAlaHisGly                              505560                                                                        LysLysValLeuThrSerLeuGlyAspAlaThrLysHisLeuAspAsp                              65707580                                                                      LeuLysGlyThrPheAlaGlnLeuSerGluLeuHisCysAspLysLeu                              859095                                                                        HisValAspProGluAsnPheLysLeuLeuGlyAsnValLeuValThr                              100105110                                                                     ValLeuAlaIleHisPheGlyLysGluPheThrProGluValGlnAla                              115120125                                                                     SerTrpGlnLysMetValThrAlaValAlaSerAlaLeuSerSerArg                              130135140                                                                     TyrHis                                                                        145                                                                           (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 146 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      ValHisPheThrAlaGluGluLysAlaAlaValThrSerLeuTrpSer                              151015                                                                        LysMetAsnValGluGluAlaGlyGlyGluAlaLeuGlyArgLeuLeu                              202530                                                                        ValValTyrProTrpThrGlnArgPhePheAspSerPheGlyAsnLeu                              354045                                                                        SerSerProSerAlaIleLeuGlyAsnProLysValLysAlaHisGly                              505560                                                                        LysLysValLeuThrSerPheGlyAspAlaIleLysAsnMetAspAsn                              65707580                                                                      LeuLysProAlaPheAlaLysLeuSerGluLeuHisCysAspLysLeu                              859095                                                                        HisValAspProGluAsnPheLysLeuLeuGlyAsnValMetValIle                              100105110                                                                     IleLeuAlaThrHisPheGlyLysGluPheThrProGluValGlnAla                              115120125                                                                     AlaTrpGlnLysLeuValSerAlaValAlaIleAlaLeuAlaHisLys                              130135140                                                                     TyrHis                                                                        145                                                                           (2) INFORMATION FOR SEQ ID NO:25:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                      MetTyrArgLeuAsnLysGluGlu                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO:26:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 141 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                      MetLeuSerProAlaAspLysThrAsnValLysAlaAlaTrpGlyLys                              151015                                                                        ValGlyAlaHisAlaGlyGluTyrGlyAlaGluAlaLeuGluArgMet                              202530                                                                        PheLeuSerPheProThrThrLysThrTyrPheProHisPheAspLeu                              354045                                                                        SerHisGlySerAlaGlnValLysGlyHisGlyLysLysValAlaAsp                              505560                                                                        AlaLeuThrAsnAlaValAlaHisValAspAspMetProAsnAlaLeu                              65707580                                                                      SerAlaLeuSerAspLeuHisAlaHisLysLeuArgValAspProVal                              859095                                                                        AsnPheLysLeuLeuSerHisCysLeuLeuValThrLeuAlaAlaHis                              100105110                                                                     LeuProAlaGluPheThrProAlaValHisAlaSerLeuAspLysPhe                              115120125                                                                     LeuAlaSerValSerThrValLeuThrSerLysTyrArg                                       130135140                                                                     (2) INFORMATION FOR SEQ ID NO:27:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 141 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                      ValLeuSerProAlaAspLysThrAsnValLysAlaAlaTrpGlyLys                              151015                                                                        ValGlyAlaHisAlaGlyGluTyrGlyAlaGluAlaLeuGluArgMet                              202530                                                                        PheLeuSerPheProThrThrLysThrTyrPheProHisPheAspLeu                              354045                                                                        SerHisGlySerAlaGlnValLysGlyHisGlyLysLysValAlaAsp                              505560                                                                        AlaLeuThrAsnAlaValAlaHisValAspAspMetProAsnAlaLeu                              65707580                                                                      SerAlaLeuSerAspLeuHisAlaHisLysLeuArgValAspProVal                              859095                                                                        AsnPheLysLeuLeuSerHisCysLeuLeuValThrLeuAlaAlaHis                              100105110                                                                     LeuProAlaGluPheThrProAlaValHisAlaSerLeuAspLysPhe                              115120125                                                                     LeuAlaSerValSerThrValLeuThrSerLysTyrArg                                       130135140                                                                     (2) INFORMATION FOR SEQ ID NO:28:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 146 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                      MetHisLeuThrProGluGluLysSerAlaValThrAlaLeuTrpGly                              151015                                                                        LysValAsnValAspGluValGlyGlyGluAlaLeuGlyArgLeuLeu                              202530                                                                        ValValTyrProTrpThrGlnArgPhePheGluSerPheGlyAspLeu                              354045                                                                        SerThrProAspAlaValMetGlyAsnProLysValLysAlaHisGly                              505560                                                                        LysLysValLeuGlyAlaPheSerAspGlyLeuAlaHisLeuAspAsn                              65707580                                                                      LeuLysGlyThrPheAlaThrLeuSerGluLeuHisCysAspLysLeu                              859095                                                                        HisValAspProGluAsnPheArgLeuLeuGlyLysValLeuValCys                              100105110                                                                     ValLeuAlaHisHisPheGlyLysGluPheThrProProValGlnAla                              115120125                                                                     AlaTyrGlnLysValValAlaGlyValAlaAsnAlaLeuAlaHisLys                              130135140                                                                     TyrHis                                                                        145                                                                           (2) INFORMATION FOR SEQ ID NO:29:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 182 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (D) OTHER INFORMATION: /note=one or both of Gly                               residues 131 and 132 can be absent; one or both                               of Gly residues 147 and 148 can be absent                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                      MetLeuSerProAlaAspLysThrAsnValLysAlaAlaTrpGlyGly                              151015                                                                        GlyTyrProTrpThrGlnArgPhePheGluSerPheGlyAspLeuSer                              202530                                                                        ThrProAspAlaValMetGlyAsnProLysValLysAlaHisGlyLys                              354045                                                                        LysValLeuGlyAlaPheSerAspGlyLeuAlaHisLeuAspAsnLeu                              505560                                                                        LysGlyThrPheAlaThrLeuSerGluLeuHisCysAspLysLeuHis                              65707580                                                                      ValAspProGluAsnPheArgLeuLeuGlyLysValLeuValCysVal                              859095                                                                        LeuAlaHisHisPheGlyLysGluPheThrProProValGlnAlaAla                              100105110                                                                     TyrGlnLysValValAlaGlyValAlaAsnAlaLeuAlaHisLysTyr                              115120125                                                                     HisGlyGlyGlyAlaAlaAlaAlaAlaAlaAlaAlaAlaAlaAlaAla                              130135140                                                                     AlaGlyGlyGlyMetHisLeuThrProGluGluLysSerAlaValThr                              145150155160                                                                  AlaLeuTrpGlyLysValAsnValAspGluValGlyGlyGluAlaLeu                              165170175                                                                     GlyArgLeuLeuValVal                                                            180                                                                           (2) INFORMATION FOR SEQ ID NO:30:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                      LeuArgArgGlnIleAspLeuGluValThrGlyLeu                                          1510                                                                          (2) INFORMATION FOR SEQ ID NO:31:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                      LysCysAlaGluLeuGluGlyLysLeuGluAlaLeuGluGlyLysLeu                              151015                                                                        GluAlaLeuGluGlyLysLeuGluAlaLeuGluGlyLysLeuGluAla                              202530                                                                        LeuGluGly                                                                     35                                                                            (2) INFORMATION FOR SEQ ID NO:32:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                      GlyGlyGlyGlyGlyGlyGly                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:33:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (D) OTHER INFORMATION: /note=one or both of Gly                               residues 2 and 3 can be absent; one or both                                   of Gly residues 17 and 18 can be absent                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                      GlyGlyGlyAlaAlaAlaAlaAlaAlaAlaAlaAlaAlaAlaAlaGly                              151015                                                                        GlyGly                                                                        (2) INFORMATION FOR SEQ ID NO:34:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (D) OTHER INFORMATION: /note=one or both of Gly                               residues 2 and 3 can be absent; any or all                                    of Pro residues 16, 17, 18 and 19 can be                                      absent; one or both of Gly residues 22 and                                    23 can be absent                                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                      GlyGlyGlyProProProProProProProProProProProProPro                              151015                                                                        ProProProGlyGlyGly                                                            20                                                                            (2) INFORMATION FOR SEQ ID NO:35:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: /note=one or both of Gly                               residues 2 and 3 can be absent; any or all                                    of Asp residues 5-33 can be absent; one                                       or both of Gly residues 35 and 36 can be                                      absent                                                                        (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                      GlyGlyGlyAspAspAspAspAspAspAspAspAspAspAspAspAsp                              151015                                                                        AspAspAspAspAspAspAspAspAspAspAspAspAspAspAspAsp                              202530                                                                        AspGlyGlyGly                                                                  35                                                                            __________________________________________________________________________

We claim:
 1. A non-naturally occurring or purified DNA moleculecomprising a DNA sequence encoding a pseudodimeric globin-likepolypeptide having two substantially homologous globin-like domains, oneof which is mutated to provide an asymmetric crosslinkable cysteineresidue, the corresponding residue in the other globin-like domain ofsaid pseudodimeric polypeptide being an amino acid other than cysteine.2. The DNA molecule of claim 1 in which the cysteine residue lies in analpha globin-like domain.
 3. The DNA molecule of claim 1 in which thecysteine residue lies in a beta globin-like domain.
 4. The DNA moleculeof claim 1 wherein each of the globin-like domains is a vertebrateglobin-like domain.
 5. The DNA molecule of claim 1 wherein each of theglobin-like domains is a mammalian globin-like domain.
 6. The DNAmolecule of claim 1 in which each of the globin-like domains is a humanglobin-like domain.
 7. The DNA molecule of claim 1, further comprising aDNA sequence encoding an additional globin-like domain bearingpolypeptide.
 8. A cell transformed with the molecule of claim 1 saidmolecule further comprising a promoter, functional in said cell, whichis operably linked to said DNA sequence, whereby said cell may be causedto express said polypeptide.
 9. A method of producing a pseudodimericglobin-like polypeptide which comprises cultivating the cell of claim 8under conditions conductive to expression of said polypeptide.
 10. Amethod of producing a pseudotetrameric hemoglobin-like protein whichcomprises cultivating the cell of claim 9, said cell further comprisingexpressible DNA sequences encoding the globin subunits of said proteinother than the globin-like domains of said polypeptide, and expressingsaid polypeptide and said subunits.
 11. A method of producing amiltimeric hemoglobin-like protein which comprises producing moleculesof a pseudotetrameric hemoglobin-like protein by the method of claim 10and then crosslinking two or more such molecules to form a molecule of amultimeric hemoglobin-like protein.
 12. The DNA molecule of claim 1wherein the globin-like domains of the pseudodimeric polypeptide aredirectly connected.
 13. The DNA molecule of claim 1 wherein theglobin-like domains of the pseudodimeric polypeptide are joined by anamino acid or peptide linker moiety.
 14. The DNA molecule of claim 1,wherein the hemoglobin-like protein is mutated, relative to humanhemoglobin, to inhibit haptoglobin binding.
 15. The DNA molecule ofclaim 14 wherein said crosslinking inhibits haptoglobin binding.
 16. TheDNA molecule of claim 1 wherein the globin-like domains of thepseudodimeric polypeptide are joined by a linker moiety consistingessentially of one or more glycines.
 17. The DNA molecule of claim 16wherein the domains are alpha globin-like domains and the linker moietyis 1-3 glycines.
 18. The DNA molecule of claim 16 wherein the domainsare beta globin-like domains and the linker moiety is 2-9 glycines. 19.The DNA molecule of claim 1, where said globin-like domains are furthermutated so that, if said pseudodimeric polypeptide is assembled into apseudotetrameric hemoglobin-like protein, said protein has a P₅₀ atleast 10% greater than does human hemoglobin Ao under the sameconditions.
 20. The DNA molecule of claim 1, where said globin-likedomains are further mutated so that, if said pseudodimeric polypeptideis assembled into a pseudotetrameric hemoglobin-like protein, saidprotein has a P₅₀ at least 10% lower than does human hemoglobin Ao underthe same conditions.
 21. The DNA molecule of claim 1, where saidglobin-like domains are further mutated so that, if said pseudodimericpolypeptide is assembled into a pseudotetrameric hemoglobin-likeprotein, said protein has a P₅₀ of 24-32 torr.
 22. The DNA molecule ofclaim 1, where said globin-like domains are further mutated so that, ifsaid pseudodimeric polypeptide is assembled into pseudotetramerichemoglobin-like protein, said protein has substantially longerintravascular retention than normal hemoglobin free in plasma.
 23. A DNAmolecule encoding a non-naturally occurring pseudooligomeric polypeptidecomprising four or more globin-like domains.
 24. The DNA molecule ofclaim 23, said polypeptide comprising, as an interdomain spacer, apolyproline helix.
 25. The DNA molecule of claim 23, said polypeptidecomprising, as an interdomain spacer, a polyaspartate or polyglutamatehelix.
 26. The DNA molecule of claim 23, said polypeptide comprising, asan interdomain spacer, an Artemia linker.
 27. The DNA molecule of claim23, said polypeptide comprising, as an interdomain spacer, a helicalcoiled coil.
 28. The DNA molecule of claim 23 wherein the globin-likedomains are human alpha globin-like domains and the interdomain spaceris about 20-50 Å.
 29. The DNA molecule of claim 28 wherein theinterdomain spacer is -(Gly)₇ (SEQ ID NO:32), -(Gly)₁₋₃ (Ala)₁₂-(Gly)₁₋₃, -(Gly)₁₋₃ (SEQ ID NO:33)(Pro)₁₂₋₁₆ -(Gly)₁₋₃ -(SEQ ID NO:34)or -(Gly)₁₋₃ (SEQ ID NO:35)(Asp)₁₋₃₀ -(Gly)₁₋₃.
 30. The DNA molecule ofclaim 23, further comprising aa DNA sequence encoding an additionalglobin-like domain bearing polypeptide.
 31. A cell transformed with themolecule of claim 23, said molecule further comprising a promoter,functional in said cell, which is operably linked to said DNA sequence,whereby said cell may be caused to express said polypeptide.
 32. Amethod of producing a pseudodimeric globin-like polypeptide whichcomprises cultivating the cell of claim 31 under conditions conducive toexpression of said polypeptide.
 33. A method of producing apseudotetrameric hemoglobin-like protein which comprises cultivating thecell of claim 32, said cell further comprising expressible DNA sequencesencoding the globin subunits of said protein other than the globin-likedomains of said polypeptide, and expressing said polypeptide and saidsubunits.
 34. A method of producing a multimeric hemoglobin-like proteinwhich comprises producing molecules of a pseudotetramerichemoglobin-like protein by the method of claim 33 and then crosslinkingtwo or more such molecules to from a molecule of a multimerichemoglobin-like protein.
 35. The DNA molecule of claim 23, saidpolypeptide being a fusion of two or more pseudodimeric polypeptidemoieties, each comprising two globin-like domains, where, in at leastone such pseudodimeric polypeptide moiety, the two globin-like domainsare directly connected.
 36. The DNA molecule of claim 23, saidpolypeptide being a fusion of two or more pseudodimeric polypeptidemoieties, each comprising two globin-like domains, where, in at leastone such pseudodimeric polypeptide moiety, the two globin-like domainsare joined by an amino acid or peptide linker moiety.
 37. The DNAmolecule of claim 23, said polypeptide being a fusion of two or morepseudodimeric polypeptide moieties, each comprising two globin-likedomains, where any two adjacent pseudodimeric polypeptide moieties areconnected by an interdomain peptide spacer, which, if saidpseudooligomeric polypeptide were incorporated into a multimerichemoglobin-like protein, would connect two pseudotetramers of saidprotein.